Coronavirus, nucleic acid, protein, and methods for the generation of vaccine, medicaments and diagnostics

ABSTRACT

A new coronavirus is disclosed herein with a tropism that includes humans. Means and methods are provided for diagnosing subjects (previously) infected with the virus. Also provided are among others vacines, medicaments, nucleic acids and specific binding members.

CROSS-REFERENCE TO RELATED APPLICATION

This patent application claims the benefit, under the provisions of 35U.S.C. §119(3), of U.S. Provisional Patent Application 60/535,002, filedJan. 7, 2004, the contents of the entirety of which is incorporatedherein by this reference.

TECHNICAL FIELD

The invention relates generally to biotechnology, especially to virologyand medicine. More particularly, the invention relates to theidentification of a new coronavirus and to means and methods associatedwith a virus such as means and methods for typing the virus in varioussamples and diagnosing of disease and means and methods for developingvaccines and medicaments for the treatment of infected subjects or ofsubjects at risk thereof.

BACKGROUND

Coronaviruses, a genus in the family of Coronaviridae, are large,enveloped plus strand RNA viruses. The genomic RNA is 27 to 32 kb insize, capped and polyadenylated. Three serologically distinct groups ofcoronaviruses have been identified. Within each group, viruses areidentified by hosts range and genome sequence. Coronaviruses have beenidentified in mice, rats, chickens, turkeys, swine, dogs; cats, rabbits,horses, cattle and humans (39, 40). Most coronaviruses infect only onehost species, and can cause severe disease including gastroenteritis,and respiratory tract diseases. In humans, 3 coronaviruses have beenstudied in detail. HCoV-229E and HCo\7-0C43 have been identified in themid sixties and are known to cause common cold (13-17 19, 41, 42).Besides common cold it has been suggested that the HCoV may cause a moreserious disease in infants as HCoV-229E virus has been isolated frominfants suffering from lower respiratory tract disease (28). The thirdand most recently identified coronavirus: SARS-CoV, is, with its abilityto cause a life threatening pneumonia (43), the most pathogenic humancoronavirus identified thus far. It has been suggested that SARS-CoV isthe first member of a fourth group of coronaviruses, or that the virusis an outlier of the group 2 coronaviruses (27, 44).

The coronavirus genome encodes four structural proteins: the spikeprotein, the membrane protein, the envelope protein and the nucleocapsidprotein. Several non-structural proteins are involved in replication andtranscription, which are encoded by two long overlapping open readingframes (ORFs) at the 5′end of the genome (1A and 1B). These 2 ORFs areconnected via a ribosomal frame shift. The polypeptides encoded by ORF1A and 1B are post-translationally processed by viral encoded proteases.Furthermore, additional non-structural proteins are encoded between theS and E gene, or between the M and N gene or downstream of the N gene.Some of these “accessory non-structural protein genes” have been foundto be not essential for virus reproduction (45, 46). The coronavirusgene products of 1A and 1B are translated from the genomic RNA but theremaining viral proteins are translated from subgenomic mRNAs (sg mRNA),each with a 5′end derived from the 5′ part of the genome. The sg mRNAare derived via a discontinuous transcription process that most probablyoccurs during negative strand synthesis (47). Discontinuoustranscription requires base-pairing between cis-acting elements, thetranscription associated sequences (TRSs), one located at the 5′ part ofthe genome (the leader TRS) and others located upstream of the OCRFs(the body TRSs) (48)).

The novel coronavirus that we present here was isolated from a childsuffering from bronchiolitis. Infection by this virus was not anisolated case since we found 7 more persons suffering from respiratorytract disease carrying the virus. In addition, we show here the completegenome sequence providing critical information concerning the genomestructure of the new coronavirus.

To date there is a range of human diseases with unknown etiology. Formany of these a viral origin has been suggested, emphasizing theimportance of a continuous search for new viruses^(22, 23, 24). Majordifficulties are encountered when searching for new viruses. First, someviruses do not replicate in vitro, at least not in the cells that arecommonly used in viral diagnostics. Second, for those viruses that doreplicate in vitro and that cause a cytopathic effect (CPE), thesubsequent virus-identification methods may fail. Antibodies raisedagainst known viruses may not recognize the cultured virus and virusspecific PCR methods may not amplify the new viral genome. We havedeveloped a method for virus discovery based on the cDNA amplifiedrestriction fragment length polymorphism technique (cDNA-AFLP). Withthis technique, RNA or DNA is reproducibly amplified. There is no needto have prior knowledge of the sequence of the target gene¹. Generallythe cDNA-AFLP method is used to monitor differential gene expression,however, we modified this method such that it can amplify viralsequences either directly from patient blood-plasma/serum samples orindirectly from CPE-positive virus culture (FIG. 1). In the modifiedVirus-Discovery-cDNA-AFLP (VIDISCA) method the mRNA isolation step priorto amplification is replaced by a treatment to selectively enrich forviral nucleic acid. Of relevance to the purification is a centrifugationstep to remove residual cells and mitochondria. In addition, a DNAsetreatment can be used to remove interfering chromosomal andmitochondrial DNA from degraded cells whereas viral nucleic acid isprotected within the viral particle. Finally, by choosing frequentlycutting restriction enzymes, the method can be fine-tuned such that mostviruses will be amplified.

In January 2003 a 7-month-old child appeared in the hospital withcoryza, conjunctivitis and fever. Chest radiography showed typicalfeatures of bronchiolitis and a nasopharyngeal aspirate specimen wascollected (sample nr: NL63) five days after the onset of disease. Alldiagnostic tests on this sample for respiratory syncytial virus (RSV),adenovirus, influenza A and B virus, parainfluenza virus type 1, 2 and3, rhinovirus, enterovirus, HCoV-229E and HCoV-OC43 were negative.Immunofluorescent assays to detect RSV, adenovirus, influenza A and Bvirus, and parainfluenza virus type 1, 2 and 3 in cultures of the virusremained negative. Acid lability and chloroform sensitivity testsdemonstrated that the virus was most likely enveloped and not a memberof the Picornavirus group. In fact it was a new coronavirus.

In the present invention we present a detailed description of a novelhuman coronavirus. Coronaviruses are characterized by a very longnon-segmented, single-stranded, (+) sense RNA of approximately 27-31 kb.This is the longest genome of any known RNA virus. The genome has a 5′methylated cap and 3′ poly-A and functions directly as mRNA. Thus faronly 3 human coronaviruses have been characterized, therefore sortingout the characteristics of a fourth human coronavirus suppliesattractive information on the variation among the human coronaviruses.The novel virus is a member of the group 1 coronaviruses and is mostrelated to HCoV-229B, yet the differences are prominent. The similarityis not larger than 85% at the nucleotide level, at the position of the4A and 4B gene of HCoV-229E only one ORF is present in HCoV-NL63 (ORF3), and the 5′ region of the S gene of HCoV-NL63 contains a unique inframe insertion of 537 nucleotides. Since binding of the receptor hasbeen mapped to the N-terminal part of the protein, the 179 amino acidsencoded by the insertion are most likely involved in receptor binding.This unique part at the N-terminus of the spike protein might explainthe expanded host range of the virus in cell culture. Where HCoV-229E isfastidious in cell culture with a narrow host range, HCoV-NL63replicates efficiently in monkey kidney cells. Besides HCoV-NL63 alsoSARS-CoV is able to replicate in monkey kidney cells (Vero-E6 cells andNCI-H292 cells for SARS-CoV (21)). Yet, comparing the predicted Spikegenes did not identify a protein region that is shared by both virusesto clarify the common host range of the viruses in vitro. Also theinsertion in the S gene of HCoV-NL63 was not present in the SARS S gene.Alternatively, other viral proteins may be involved in the cell tropismof a virus, however we did not identify any gene of HCoV-NL63 that hadmore similarity at the protein level to the SARS-CoV than to thesimilarity to HCoV-229E.

The 2 major differences between HCoV-229E and HCoV-NL63: the insertionin the S gene and the altered non-structural accessory proteins genes,are comparably to the differences that are noted between the porcinecoronaviruses PRCoV and TGEV. Although these 2 porcine viruses areantigenically and genetically related their pathogenicity is verydifferent. TGEV causes severe diarrhea with a high mortality in neonatalswine. It replicates and destroys the enterocytes in the small intestinewhereas PRCoV has a selective tropism for respiratory tissue with verylittle to no replication in intestinal tissue. The genome differences inthe S, 3A and 3B genes between TGEV and PRCoV are comparable with thedifferences between HCoV-NL63 and HCoV-229E. Alike HCoV-NL63, TGEV has aunique in frame insertion at the 5′ part of the S gene ranging from 672to 681 nt (53). Furthermore, the accessory protein genes 3A and 3B thatare intact in TGEV, are often mutated or inactive in the PRCoV.Extrapolating these data to the human coronaviruses one can speculatethat HCoV-NL63 might be a more pathogenic human virus in comparison withHCoV-229E. However there are no epidemiological data supporting this.Based on our data it seems likely that HCoV-NL63 and HCoV-229E share thesame pathogenicity. The common cold virus HCoV-229E can cause a moreserious disease in infants (28), comparable to our data that suggestthat HCoV-NL63 is causing a respiratory disease only in infants andimmuno-compromised patients.

To date, a viral pathogen cannot be identified in a substantial portionof respiratory disease cases in humans (on average 20%⁵⁹), our dataindicate that in a part of these cases HCoV-NL63 is involved. Thefrequency with which HCoV-NL63 was detected in patients suffering fromrespiratory disease was up to 5% in January 2003. The virus was notdetected in any of the samples collected in the spring or summer of2003, which is in harmony with the epidemiology of human coronavirusesthat have a tendency to spread predominantly in the winter season (15).The primers for our diagnostic PCR were located in the 1B gene and thegenomic RNA can be used as template. Using primers that anneal in thenucleocapsid gene or 3′UTR supplies more template in the PCR becausebesides the genomic RNA also all sg mRNA in infected cells are templatefor amplification. It might be that the number of persons that we foundpositive for HCoV-NL63 is an underestimation of the correct number ofpersons carrying HCoV-NL63.

The newly found coronavirus, (designated HCoV-NL63) was characterizedand sequenced. A sequence of a prototype HCoV-NL63 is provided in FIG.19 and parts thereof in table 3. In one aspect the invention thereforeprovides an isolated and/or recombinant nucleic acid comprising asequence as depicted in FIG. 19 and/or table 3, or a functional part,derivative and/or analogue thereof. The virus HCoV-NL63 is characterizedby the prototype, however, many natural variants exist as for instanceshown in FIG. 16 for polymorphisms in the ORF 1a region. The existenceof such natural variants is normal for RNA viruses that undergo frequentmutation through for instance the introduction of mistakes by thepolymerases that copy the genome. HCoV-NL63 viruses that have a slightlydivergent nucleic acid sequence are thus also provided by the presentinvention. Such viruses are considered to be a derivative of the nucleicacid having the prototype nucleic acid sequence. The variant does notnecessarily have to be a natural variant. It is very well possible togenerate variants through recombinant means. For instance many parts ofthe virus can be altered through nucleotide substitution to make use ofthe redundancy in the triplet genetic code for particular amino acids.Thus without altering the amino acid sequence of the encoded proteins.However, even amino acid alterations can typically be introduced withoutaffecting the replicating and coding potential of the viruses. Forinstance conservative amino acid substitutions are often tolerated.Alterations in the prototype virus may be up to 70% of the nucleic acidsequence without altering the replicating potential of the virus. Thusin one embodiment the invention provides an isolated and/or recombinantnucleic acid that is at least 70% homologous to a nucleic acid of theprototype RCoV-NL63. Most of the viable variants however are at least95% homologous and more preferably at least 99% to a nucleic acidaccording to the prototype HCoV-NL63. The homology between differentcoronaviruses in the UTR regions is typically high, for this reason thehomology in this application is measured in a region outside the UTRregions, preferably in a protein coding region. Thus the inventionprovides a derivative of HCoV-NL63 virus comprising at least 95%homology and preferably at least 99% homology (on the nucleic acidlevel) in at least one protein coding region depicted FIG. 20, 21, 22,23, or table 3. The nucleic acid of the virus or parts thereof can becloned and used as a probe to detect the virus in samples. Thus thepresent invention further provides an isolated and/or recombinantnucleic acid comprising a stretch of 100 consecutive nucleotides of anucleic acid of the prototype virus, or a region that is at least 95%and preferably at least 99% homologous to said 100 consecutivenucleotides(when measured on the nucleic acid level outside a UTRregion). A stretch of 100 consecutive nucleotides is considered to be afunctional part of the virus of the present invention. Further providedis a bacterial vector comprising a nucleic acid of HCoV-NL63 or afunctional part, derivative and/or analogue thereof. Further provided isa bacterium comprising said bacterial vector. The sequence of HCoV-NL63or a part thereof can be used to generate a primer that is specific forHCoV-NL63 and thus capable of specifically replicating HCoV-NL63 nucleicacid. Similarly, a probe can be generated that specifically hybridizesto HCoV-NL63 nucleic acid under stringent conditions. Thus the inventionfurther provides a primer and/or probe, capable of specificallyhybridizing to a nucleic acid of a HCoV-NL63 virus or functional part,derivative or analogue thereof. Preferably, said primer or probe iscapable of hybridizing to said nucleic acid under stringent conditions.In a particularly preferred embodiment said primer and/or probecomprises a sequence as depicted in table 3, table 7, table 10 or FIGS.16 to 18. The nucleic acid of the prototype virus encodes variousproteins and poly-proteins. These proteins are expressed for instance incells producing the virus or transformed with a nucleic acid encodingthe (poly)protein. The invention thus further provides an isolatedand/or recombinant proteinaceous molecule comprising a sequence asdepicted in FIG. 20, 21, 22, 23 or table 8, or a functional part,derivative and/or analogue thereof. Many different variants of theproteins having the same function in kind, not necessarily in amountare, as mentioned above, present in nature and can be generatedartificially, thus the invention further provides an isolated and/orrecombinant proteinaceous molecule that is at least 70% homologues to aproteinaceous molecule mentioned above. Such homologous proteins areconsidered derivatives of a protein encoded by the prototype.Preferably, a derivative protein comprises at least 95% and morepreferably at least 99% homology with a protein encoded by the prototypeHCoV-NL63. Fragments and parts of a proteinaceous molecule encoded bythe prototype virus can be generated, such parts are therefore alsoprovided by the present invention. In a preferred embodiment is providedan isolated and/or recombinant proteinaceous molecule comprising astretch of at least 30 consecutive amino acids of a proteinaceousmolecule encoded by the prototype virus. A protein encoded by theprototype virus can be encoded through a variety of different nucleicacid sequences using the redundancy of the genetic code. Thus theinvention further provides a nucleic acid encoding a protein depicted inFIG. 20, 21, 22, 28 or table 3. The HCoV-NL63 virus can be replicatedusing in vitro growing cell lines. The virus can be harvested from suchcultures and used in a variety of different application including butnot limited to the generation of an immune response in a subject. Theinvention thus further provides an isolated or recombinant viruscomprising a HCoV-NL63 nucleic acid sequence or a functional part,derivative and/or analogue thereof. Also provided is an isolated orrecombinant virus comprising a proteinaceous molecule as depicted inFIG. 20, 21, 22, 23 or table 8, or a functional part, derivative and/oranalogue thereof. Subjects that have become infected with HCoV-NL63 candisplay a number of different clinical and/or subclinical symptoms. Thusfurther provided is an isolated or recombinant virus or a functionalpart, derivative or analogue thereof capable of inducing aHCoV-N63-related disease. The virus comprises substances that can beused to generate specific binding partners that are able to specificallybind the substance of the virus. Binding partners can be generated bymeans of injection of the virus into in an immuno-competent subject. Asa result of the immunization the serum obtained from the subject willtypically contain a number of different antibodies specific for thevirus or an immunogenic part, derivative and/or analogue thereof.Specific binding partners can of course be generated through a largevariety of different technologies. For instance phage displaytechnologies. The method of producing the specific binding partner isnot limited herein. The binding is typically specific for aproteinaceous part of the virus. But can of course also be specific fora virus specific post translation modification of a protein contained inthe virus. Thus the present invention further provides an isolatedbinding molecule capable of specifically binding a proteinaceousmolecule of a HCoV-NL63 virus preferably against encoded by a nucleicacid of the prototype HCoV-NL63. Preferably, a proteinaceous molecule asdepicted in FIG. 20, 21, 22, 23 or table 3, or a functional part,derivative and/or analogue thereof. The binding molecule can be capableof specifically binding a nucleic acid sequence of a HCoV-NL63,preferably of FIG. 19 or table 3. The binding molecule is preferably aproteinaceous molecule. However, other binding molecules are also withinthe scope of the present invention. For instance, it is possible togenerate protein mimetics or analogues having the same binding qualityas a protein in kind not necessarily in amount. Provided is further amethod for producing a binding molecule according to the inventioncomprising

-   -   producing molecules capable of binding a HCoV-NL63 virus or        functional part, derivative or analogue thereof or an isolated        and/or recombinant proteinaceous molecule encoded by a prototype        nucleic acid of HCoV-NL63, and    -   selecting a proteinaceous binding molecule that is specific for        said virus and/or said proteinaceous molecule.

The overall homology of HCoV-NL63 virus with other human coronavirusesis not very high. Thus many different binding molecules capable ofspecifically binding to HCoV-NL63 virus can be generated. Such bindingmolecules can be used to detect HCoV-NL63 virus in a sample. Theinvention thus further provides an isolated or recombinant virus whichis immunoreactive with a binding molecule capable of specificallybinding HCoV-NL63 virus. Similarly, the invention provides the use of anisolated and/or recombinant proteinaceous molecule as depicted in FIG.20, 21, 22, 23 or table 3, or a functional part, derivative and/oranalogue thereof, for detecting a binding molecule capable ofspecifically binding HCoV-NL63 virus, or functional part, derivativeand/or analogue of said virus in a sample Vise versa, HCoV-NL63 viruscan be used to detect a molecule capable of specifically binding saidvirus in a sample. Binding of HCoV-NL63 virus to a susceptible targetcell occurs via a specific receptor. This receptor can be used as abinding molecule of the invention. Preferably, the binding moleculecomprises an antibody or functional equivalent thereof. The detectionmethods can be used to diagnose HCoV-NL63 related disease in a subject.Thus provided is a method for detecting a HCoV-NL63 virus or functionalpart, derivative or analogue thereof in a sample, comprising hybridizingand/or amplifying a nucleic acid of said virus or functional part,derivative or analogue with a HCoV-NL63 specific primer and/or probe anddetecting hybridized and/or amplified product. Further provided is akit, preferably a diagnostic kit comprising a HCoV-NL63 virus orfunctional part, derivative or analogue thereof a binding moleculeaccording to the invention, and/or a HCoV-NL63 virus specificprimer/probe according to invention.

In a particular preferred embodiment is provided the use of a primer orprobe capable of specifically hybridizing to a nucleic acid of aHCoV-NL63 virus or functional part, derivative or analogue thereof or abinding molecule capable of specifically binding a proteinaceousmolecule depicted in FIG. 20, 21, 22, 23 or table 3 or an HCoV-NL63virus and/or a nucleic acid or functional part, derivative or analogueof a prototype HCoV-NL63 for detecting and/or identifying a HCoV-NL63coronavirus in a sample. Preferably said nucleic acid comprises asequence as depicted in table 3.

The invention further provides a vaccine comprising HCoV-NL63 virus orfunctional part, derivative or analogue thereof. Further provided is avaccine comprising a proteinaceous molecule depicted in FIG. 20, 21, 22,23 or table 3 or functional part, derivative and/or analogue of such aproteinaceous molecule. A proteinaceous molecule of the invention may beprovided as a vaccine by itself or as a part of the protein or asderivatives or analogues thereof. A suitable analogue is a nucleic acidencoding a HCoV-NL63 virus proteinaceous molecule or a functional partor derivative thereof. The nucleic acid may be used in a DNA vaccineapproach which is also provided in the present invention. As carrier forthe DNA vaccine it is often suitable to incorporate an expressibleHCoV-NL63 virus nucleic acid in a viral replicon allowing replication ofthe HCoV-NL63 virus nucleic acid in the target cell and thereby allowingboosting of the provided immune response. A HCoV-NL63 virus encodedprotein that is suited for such a DNA vaccine approach is the S proteindepicted in FIG. 22 or a functional part, derivative and/or analoguethereof. A part of an S protein preferably comprises an immunogenic partof the 537 in frame insertion as compared with HCoV-229E virus.Preferably said part comprises essentially said 537 insertion. With the537 insertion is meant a sequence corresponding to sequences 20472 to21009 of FIG. 19. Other suitable candidates are the M and or the Nprotein or a functional part, derivative and/or analogue thereof.Typically a vaccine includes an appropriate adjuvant. Apart from the usein a vaccine the mentioned virus and/or proteinaceous molecules can alsobe used to generate and/or boost a HCoV-NL63 virus specific immuneresponse in a subject. The immune response can be both cellular orhumoral. Thus further provided is an isolated T-cell comprising a T-cellreceptor that is specific for HCoV-NL63 virus or a proteinaceousmolecule encoded by a prototype HCoV-NL63 virus. Further provided is anisolated B-cell producing an antibody specific for HCoV-NL63 virus or aproteinaceous molecule encoded by a HCoV-NL63 virus. The antibody orT-cell receptor can be cloned whereupon a cell line can be provided withan expression cassette comprising the cloned receptor or antibody. Thusthe invention further provides a cell producing such a receptor orantibody. Such a cell is preferably a cell that is suitable for largescale production of the mentioned proteins such as CHO cells.

It is also possible to provide a subject with passive immunity toHCoV-NL63 virus. To this end the subject can be provided with aHCoV-NL63 specific binding molecule of the invention. Such immunity canbe used to provide a barrier for (further) infection with HCoV-NL63virus in the subject, thus further provided is a vaccine comprising aHCoV-NL63 virus specific binding molecule according to the invention. Ina preferred embodiment, passive immunity is provided by a human orhumanized antibody capable of specifically binding a HCoV-NL63 virus ofthe invention. The barrier does not have to be perfect. The presence ofa binding molecule at least reduces the spread of the virus to othertarget cells in the subject. The passive immunity may be administered toa subject as prophylactic to at least reduce the spread of HCoV-NL63virus in the subject when exposed to the virus. Alternatively, thepassive immunity may be provided to a subject already infected with thevirus. In the latter case one or more HCoV-NL63 virus specific bindingmolecules of the invention are used as a medicament to at least reducethe spread of the virus in the subject and thereby at least in partcombat the virus infection. The invention thus further provides amedicament comprising a HCoV-NL63 virus specific binding moleculeaccording to the invention. Further provided is the use of a virus ofthe invention or functional part, derivative or analogue thereof or aproteinaceous molecule of the invention or a HCoV-NL63 virus specificbinding molecule of the invention, for the preparation of a vaccineagainst a coronaviral genus related disease. Further provided is amethod for treating an individual suffering from, or at risk ofsuffering from, an HCoV-NL63 related disease, comprising administeringto said individual a vaccine or medicament according to the invention.In yet another embodiment is provided a method for determining whetheran individual suffers from an HCoV-NL63 related disease, comprisingobtaining a sample from said individual and detecting a HCoV-NL63 virusor functional part, derivative or analogue thereof in said sample.

In yet another embodiment is provided an isolated cell, or recombinantor cell line comprising HCoV-NL63 virus, or a functional part,derivative and/or analogue thereof. Preferably said cell is a primatecell, preferably a monkey cell. In a preferred embodiment, said cell isa cell that replicates the HCoV-NL63 virus of the invention. In aparticular embodiment the cell is a kidney cell. The cell can be used toproduce the HCoV-NL63 virus of the invention or to attenuate HCoV-NL63such that it becomes less pathogenic. Virus attenuation is spontaneousupon continued culture of the virus on the mentioned preferred celllines. Attenuated HCoV-NL63 virus can be used as a vaccine.

HCoV-NL63 virus encodes an endoprotease. A sequence for the protease inthe prototype

HCoV-NL63 virus is depicted in FIG. (21). The protease is important forthe processing of the polyproteins encoded by HCoV-NL63. The action ofthe protease is at least in part inhibited by a viral protease inhibitoras further described herein. Thus the invention further provides acompound for at least in part inhibiting HCoV-NL63 virus replication.Preferred compounds are inhibitors of inosine monophosphatedehydrogenase (55) (e.g. Ribavirin(54) and mycophenolic acid),orotidine-5′-phosphate decarboxylase inhibitors (e.g. 6-azauridine andpyrazofurin), 3CL-protease inhibitors(56) (e.g. the VNSTLQ (SEQ. ID. NO:1)-AG7088 ester, see below), cap-methylase inhibitors(58) (carboxylicadenosine analogs e.g. Neoplanocin A and 3-deazaneoplancin A), nitrousoxide synthase inducing compounds (e.g. glycyrrhizin) and Interferons(57). Of these the protease inhibitors are particularly preferred. Thesequence VNSTLQ (SEQ. ID. NO: 1) is the N-terminal proteolyticprocessing site of SARS-3CLpro that is used in the 3Clpro inhibitorVNSTLQ (SEQ. ID. NO: 1)-AG7088 (56). In this compound the hexapeptideVNSTLQ (SEQ. ID. NO: 1) is C-terminally linked to the vinylogous ethylester (AG7088, see structural formula 1 depicted below,) that inhibitsSARS 3CLpro activity.

The hexapeptide VNSTLQ (SEQ. ID. NO: 1) corresponds to YNSTLQ (SEQ. ID.NO: 2)in HCoV-NL63. Therefore YNSTLQ (SEQ. ID. NO: 2)- AG7088 inhibitsthe HCoV-NL63 3CLpro orthologs. Thus in a preferred embodiment theprotease inhibitor comprises the amino acid sequence VNSTLQ (SEQ. ID.NO: 1) more preferably YNSTLQ (SEQ. ID. NO: 2). Analogues of suchprotease inhibitors that comprise the same activity in kind notnecessarily in amount are also provided by the present invention. Suchanalogues include, compounds comprising a peptide with the preferredsequence, wherein the peptide comprises a modification. Other analoguesinclude compounds having protein mimetic activity that mimic thepreferred amino-acid sequence. S-adenosylmethionine-dependant ribose2′-orthomethyltransferase Plays a role in the methylation of capstructure (GpppNm) at the 5′end of the viral RNA. Antiviral compoundsinhibiting this transfer of methyl groups to reaction (carboxylicadenosine analogs e.g. Neoplanocin A and 3-deazaneoplancin A) interferewith expression of viral proteins.

The invention further provides a proteinaceous molecule encoded byHCoV-NL63 nucleic acid, wherein said proteinaceous molecule is a 3CLprotease or a functional equivalent thereof. Functional equivalentsinclude an proteolytically active part and/or derivative having one ormore conservative amino acid substitutions. There are many methods knownin the art to determine whether a compound has anticoronaviral activity,preferably antiproteolytic activity of a coronavirus. The invention thusfurther provides a method for determining whether a compound comprisesanticoronavirus replication activity characterized in that said methodutilizes HCoV-NL63-virus or a HCoV-NL63 protein involved in replicationof HCoV-NL63 or a functional part, derivative and/or analogue thereof.Preferably, the invention provides a method for determining whether acompound is capable of at least in part inhibiting a viral proteasecharacterized in that said protease is a 3CL protease of HCoV-NL63 or afunctional part, derivative and/or analogue thereof. Preferred compoundsthat can be tested for 3CL inhibiting quality are hexapeptides locatedN-terminally of 3Clpro cleavage sites. Compounds effective in at leastin part inhibiting 3Cl proteolytic activity can be used for thepreparation of a medicament for the treatment of an individual sufferingor at risk of suffering from a HCoV-NL63 virus infection.

One or more of the preferred anticoronaviral replication compounds canbe used as a medicament for the treatment of a subject suffering from orat risk of suffering from a HCoV-NL63 virus infection. The inventionthus further provides a medicament for the treatment of an individualsuffering from an coronavirus infection or an individual at risk ofsuffering there from comprising wherein said coronavirus comprises anucleic acid sequence of a HCoV-NL63 prototype virus or a functionalpart, derivative and/or analogue thereof.

In the present invention several different recombinant viruses areproduced using HCoV-NL63 virus nucleic acid as a backbone. Suchreplication competent or replication defective recombinant virus can beused for instance as gene delivery vehicles. On the other hand parts ofa HCoV-NL63 virus can be used in gene delivery vehicles that are basedon other means for delivering genetic material to a cell. Thus theinvention further provides a gene delivery vehicle comprising at leastpart of a HCoV-NL63 virus nucleic acid. Preferably of the prototypevirus. Preferably comprising a nucleic acid encoding a protein ofHCoV-NL63 virus or a functional part, derivative and/or analoguethereof. The invention also shows chimearic coronaviruses comprisingnucleic acid derived from at least two coronaviruses wherein at leastone of said parts is derived from a HCoV-NL63 virus. Said HCoV-NL63virus derived part comprises preferably at least 50 nucleotides of aprotein coding domain. More preferably said HCoV-NL63 derived partcomprises at least 500 and more preferably at least 1000 nucleotides ofthe sequence as depicted in FIG. 19 or a functional derivative thereof.In a preferred embodiment the invention provides a chimearic coronaviruscomprising at least 1000 nucleotides of a sequence as depicted in FIG.19 and at least 1000 nucleotides of another coronavirus wherein saidlatter 1000 nucleotides comprise a sequence that is more than 5%sequence divergent with a sequence as depicted in FIG. 19. The sequencesof a number of HCoV-NL63 virus fragments are depicted in table 3. Thelocation of the fragments in the large genomic RNA is depicted in FIG.5. The invention therefore, in one aspect, provides an isolated orrecombinant virus comprising a nucleic acid sequence as depicted intable 3, or a functional part, derivative or analogue of said virus.With the aid of the identifying prototype fragments it is possible tofurther sequence the genome. One way of doing this by primer walking onthe genome. A primer is directed to a region of which the sequence isknown and this primer is used to sequence a flaking region that is asyet unknown. A subsequent primer can be generated against the newlyidentified sequence and a further region can be sequenced. Thisprocedure can be repeated until the entire sequence of the virus iselucidated. As a source of the virus one may turn to Dr. C. van derHoek, Department of Human Retrovirology, Academic Medical Center,University of Amsterdam, Amsterdam, The Netherlands.

Alignments of the determined nucleic acid sequences revealed the readingframe used in the sequences found, accordingly the invention furtherprovides an isolated or recombinant virus comprising an amino acidsequence as depicted in (table 3). or a functional part, derivative oranalogue of said virus. A particular amino acid sequence can be producedfrom a variety of nucleic acids depending on the codons used. Thus theinvention further provides a nucleic acid encoding an amino acidsequence as depicted in (table 3). Further provided is an isolated orrecombinant virus comprising a nucleic acid sequence encoding an aminoacid sequence as depicted in (table 3 ), or a functional part,derivative or analogue of said virus.

Coronaviruses as many other types of viruses acquire a plurality ofspontaneous and selected mutations upon spreading of the virus throughthe subject population and/or during culturing ex vivo. Moreover,artificial mutations having no recognized counterpart in nature can beintroduced into the sequence of the prototype virus or a derivativethereof, without altering the viral- and/or disease causing propertiesof the virus. Having characterized the prototype of the newly discoveredsubtype gives access to this group of viruses belonging to the samesubtype. Thus the invention further provides an isolated or recombinantvirus comprising a nucleic acid sequence that is approximately 80%homologous to a sequence as depicted in table 3, or 80% homologous to anamino acid sequence depicted in Table 3 (. Preferably the homology is atleast 90%, more preferably at least 95% and even more preferably atleast 99%.

The respective prototype fragments were compared with a database ofviral sequences and hits having a particularly high homology arementioned in the tables 5 and 6. It may be noted that the comparedfragments do not share extensive homology with any of the currentlyknown Coronaviruses. The invention thus provides an isolated and/orrecombinant virus comprising an amino acid sequence which is more than89% homologous to 163-2 amino acid sequence as depicted in Table 3.Preferably said homology is at least 90%, more preferably at least 95%and even more preferably at least 99%. Further provided is an isolatedor recombinant virus comprising an amino acid sequence which is morethan 60% homologous to 163-4 amino acid sequence as depicted in Table 3.Preferably said homology is at least 90%, more preferably at least 95%and even more preferably at least 99%. Further provided is an isolatedor recombinant virus comprising a nucleic acid sequence which is morethan 85% homologous to 163-9 nucleic acid sequence as depicted in Table3. Preferably said homology is at least 90%, more preferably at least95% and even more preferably at least 99%. Further provided is anisolated or recombinant virus comprising an amino acid sequence which ismore than 94% homologous to 163-10 amino acid sequence as depicted inTable 3. Preferably said homology is at least 90%, more preferably atleast 95% and even more preferably at least 99%. Further provided is anisolated or recombinant virus comprising an amino acid sequence which ismore than 50% homologous to 163-11 amino acid sequence as depicted inTable 3. Preferably said homology is at least 90%, more preferably atleast 95% and even more preferably at least 99%.

Further provided is an isolated or recombinant virus comprising an aminoacid sequence which is more than 87% homologous to 163-14 amino acidsequence as depicted in Table 3. Preferably said homology is at least90%, more preferably at least 95% and even more preferably at least 99%.Further provided is an isolated or recombinant virus comprising an aminoacid sequence which is more than 88% homologous to 163-15 amino acidsequence as depicted in Table 3. Preferably said homology is at least90%, more preferably at least 95% and even more preferably at least 99%.Further provided is an isolated or recombinant virus comprising an aminoacid sequence which is more than 78% homologous to 163-18 amino acidsequence as depicted in Table 3. Preferably said homology is at least90%, more preferably at least 95% and even more preferably at least 99%.Further provided is an isolated or recombinant virus comprising anucleic acid sequence which is at least 50% homologous to a nucleic acidsequence as depicted in Table 3. Preferably said homology is at least80%, more preferably at least 90%, more preferably at least 95% and evenmore preferably at least 99%.

The invention also provides a functional part, derivative and/oranalogue of an isolated and/or recombinant HCoV-NL63 virus. A part of avirus can be a membrane containing part, a nucleocapsid containing part,a proteinaceous fragment and/or a nucleic acid containing part. Thefunctionality of the part varies with the application chosen for thepart, for instance, part of the virus may be used for immunizationpurposes. In this embodiment the functionality comprises similarimmunogenic properties in kind as the entire virus not necessarily inamount. Another use of the virus is the infectivity of the virus, forinstance, for in vitro (or in vivo) culture, in this embodiment thefunctionality comprises a similar infectivity in kind not necessarily inamount. Many other functionalities may be defined, as there are manydifferent uses for viruses, non-limiting examples are the generation ofchimeric viruses, (i.e. with one or more other (corona) viruses, and thegeneration of viral vectors for vaccination and/or gene therapeuticpurposes. Such viruses and/or vectors also contain a functional part ofHCoV-NL63 and are thus also encompassed in the present invention. Afunctional derivative of a virus of the invention is defined as a virusthat has been altered such that the properties of said compound areessentially the same in kind, not necessarily in amount. A derivativecan be provided in many ways, for instance through nucleotidesubstitution (preferably “wobble” based), through (conservative) aminoacid substitution, subsequent modification, etcetera.

Analogous compounds of a virus can also be generated using methods inthe art. For instance, a chimeric virus can be produced, or an HCoV-NL63virus having a chimeric protein. For instance, HCoV-NL63 can be renderedmore immunogenic by generating a cell surface associated fusion proteincomprising at least part of an HCoV-NL63 surface protein and anon-HCoV-NL63 immunogenic part. HCoV-NL63 virus comprising such chimericprotein can be used for inducing an enhanced immune response in a host,for instance for vaccination purposes.

As used herein, the term “a virus of the invention” is meant to alsocomprise a functional part, derivative and/or analogue of said virus.

The three groups of coronaviruses are associated with a variety ofdiseases of humans and domestic animals, including gastroenteritis andupper and lower respiratory tract disease. The human coronavirusesHCoV-229E and HCoV-OC43 are associated with mild disease (the commoncold) but more severe disease is observed in children¹⁶, albeit at avery low incidence. Several coronaviruses cause a severe disease inanimals and SARS-CoV is the first example of a coronavirus that causessevere disease in humans. However, it should be emphasized that asubstantial part of respiratory disease cases in humans remainsundiagnosed. For instance, a recent survey of respiratory viruses inhospitalized children with bronchiolitis in Canada could not reveal aviral pathogen in about 20% of the patients¹⁷. The fact that weidentified the new coronavirus in a child with bronchiolitis shows thatHCoV-NL63 is a pathogenic respiratory virus. When considering that theHCoV-NL63 is a pathogenic respiratory virus able to cause bronchiolitisin infected children, the interesting question remains why HCoV-NL63 wasnot recognized previously by cell culture. We found that the virus canbe cultured in monkey kidney cells (tMK or LLC-MK2 cells), cells thatare often used in a routine diagnostic setting and one might thereforespeculate that HCoV-NL63, like SARS-CoV, was newly introduced from ananimal reservoir into the human population or that this is a human virusthat recently broadened its host cell range. Clearly it is of importanceto study the prevalence of HCoV-NL63 infection, and screening specimensfrom patients with respiratory tract disease using the HCoV-NL63diagnostic RT-PCR will shed light on this issue. It is remarkable thatthe new human coronavirus was harvested from tMK cells and LLC-MK2 cellssince coronaviruses are typically fastidious in cell culture with anarrow host range. However, both SARS-CoV and HCoV-NL63 seem toreplicate efficiently in monkey kidney cells (Vero-E6 cells and NCI-H292cells for SARS-CoV). The recently described genome of SARS-CoV hasseveral exclusive features, including some unique open reading framesthat are probably of biological significance^(15, 18). We will thereforeanalyze the complete genome sequence of HCoV-NL63 to screen forsimilarities and differences with SARS-CoV that may determine theexpanded host cell range and enhanced pathogenicity of these viruses.

HCoV-NL63 is associated with a particular phenotype in infectedsubjects. The phenotype can encompass bronchiolitis, coryza,conjunctivitis and fever and may further encompass other respiratoryproblems and diarrhea. In one embodiment the invention thus furtherprovides an isolated and or recombinant virus of the invention (havingone or more of the above mentioned homology) wherein said virus orfunctional part, derivative and/or analogue further comprises thecapability to induce an HCoV-NL63 related disease or symptom in asubject. In another embodiment the invention provides an isolated and/orrecombinant virus of the invention further comprising the property tocause CPE in tertiary monkey kidney cells (tMK; Cynomolgus monkey³⁷)and/or upon passage onto the monkey cell line LLC-MK2 (ECCAC 85062804,ATCC CCL-7). In a preferred embodiment said virus does not produce CPEin Vero-cells (ATCC CRL-1586)³⁴.

The invention further provides a nucleic acid as depicted in table 3,and an amino acid sequence as depicted in Table 3, or a functional partand/or equivalent of such a nucleic acid and/or amino acid sequence. Afunctional equivalent of said nucleic acid comprises the samehybridization properties in kind, not necessarily in amount, as saidnucleic acid (or part thereof). A functional equivalent of an amino acidsequence of the invention comprises the same immunogenic properties inkind, not necessarily in amount, as said amino acid sequence (or partthereof). A part of a nucleic acid of the invention comprises at least15 nucleotides, preferably at least 20, more preferably at least 30nucleotides. A part of an amino acid sequence comprises at least 5 aminoacids in peptidic linkage with each other, more preferably at least 8,and more preferably at least 12, more preferably at least 16 aminoacids. In a preferred embodiment said nucleotides and/or amino acids areat least semi-consecutive, more preferably, said nucleotides and/oramino acids are consecutive. An equivalent of a nucleic acid and/oramino acid sequence of the invention or part thereof comprises at least80% homology to a nucleic acid and/or amino acid sequence of theinvention, preferably at least 90% homology, more preferably at least95% and even more preferably at least 99% homology to a nucleic acidand/or amino acid sequence of the invention or a part thereof.

The invention further provides a primer and/or probe, capable ofspecifically hybridizing to a nucleic acid of a virus or functionalpart, derivative or analogue according to the invention, preferably aprimer and/or probe, capable of specifically hybridizing to a nucleicacid sequence as depicted in Table 3. More preferably, a primer and/orprobe, which is capable of hybridizing to said nucleic acid understringent conditions. In a particular preferred embodiment is provided aprimer and/or probe, comprising a sequence as depicted in Table 7.

The art knows many ways in which a specific binding member can begenerated against an identified nucleic acid, lipid and/or amino acidsequence. Such specific binding members may be of any nature but aretypically of a nucleic acid and/or proteinaceous nature. The inventionthus further provides an isolated molecule capable of specificallybinding a virus, nucleic acid and/or amino acid or functional part,derivative or analogue thereof according to the invention. Said isolatedmolecule is also referred to as specific binding member. Preferably saidspecific binding member is capable of specifically binding at least partof a nucleic acid sequence as depicted in table 8 and/or at least partof an amino acid sequence as depicted in Table 3. In a preferredembodiment said binding member is a proteinaceous molecule. Preferablyan antibody or a functional part, derivative and/or analogue thereof. Aspecific binding member preferably comprises a significantly betterbinding property for the HCoV-NL63 virus compared to unrelated control.However, for instance for antibodies, it is possible that the epitopespecifically recognized in HCoV-NL63 is also present in a limited numberof other molecules. Thus though the binding of the binding member may bespecific, it may recognize also other molecules than those present inHCoV-NL63. This cross-reactivity is to be separated from a-specificbinding and is a general property of antibodies. Cross-reactivity doesnot usually hinder the selection of suitable specific binding membersfor particular purposes. For instance a specific binding member thatalso recognized a protein in liver cells can be used in manyapplications even in the presence of liver cells, where additionalinformation such as location in the cell can often be used todiscriminate.

One source of an antibody of the invention is the blood of the infectedsubjects screened for the virus of the present invention. One mayfurther characterize B-cells obtained from said subject, A suitableB-cell may be cultured and the antibody collected. Alternatively, theantibody may be sequenced from this B-cell and generated artificially.Another source of an antibody of the invention can be generated byimmunisation of test animals or using artificial libraries to screen apurified fraction of virus. A functional part of an antibody hasessentially the same properties of said antibody in kind, notnecessarily in amount. Said functional part is preferably capable ofspecifically binding an antigen of HCoV-NL63. However, said functionalpart may bind such antigen to a different extend as compared to saidwhole antibody. A functional part or derivative of an antibody forinstance comprises a FAB fragment or a single chain antibody. Ananalogue of an antibody for instance comprises a chimeric antibody. Asused herein, the term “antibody” is also meant to comprise a functionalpart, derivative and/or analogue of said antibody.

Once antibody of the invention is obtained, a desired property, such asits binding capacity, can be improved. This can for instance be done byan Ala-scan and/or replacement net mapping method. With these methods,many different proteinaceous molecules are generated, based on anoriginal amino acid sequence but each molecule containing a substitutionof at least one amino acid residue. Said amino acid residue may eitherbe replaced by Alanine (Ala-scan) or by any other amino acid residue(replacement net mapping). Each variant is subsequently screened forsaid desired property. Generated data are used to design an improvedproteinaceous molecule.

There are many different ways in which a specific binding member can begenerated. In a preferred embodiment the invention provides a method forproducing a specific proteinaceous binding member comprising producingproteinaceous molecules capable of binding a virus according to theinvention or to a functional part, derivative or analogue, and selectinga proteinaceous molecule that is specific for said virus. If need be,the method may be used to generate a collection of proteinaceousmolecules capable of binding to said virus or functional part,derivative and/or analogue thereof and selecting from said collectionone or more binding members capable of specifically binding said virusor functional part, derivative and/or analogue thereof.

Any specific binding member is characteristic for the HCoV-NL63 virus ofthe invention. Thus a virus that is specifically reactive with suchbinding member is an HCoV-NL63 virus and thus provided by the invention.Thus the invention provides an isolated and/or recombinant virus that isimmunoreactive with specific binding member of the invention, preferablya proteinaceous binding member. The invention further provides acomposition of matter comprising isolated HCoV-NL63 virus, and/or avirus essentially corresponding to HCoV-NL63. The term, a virus“essentially corresponding to HCoV-NL63” refers to HCoV-NL63 viruseswhich are either identical to the HCoV-NL63 strain describedhereinabove, or which comprises one or more mutations compared to thesaid HCoV-NL63strain. These mutations may include natural mutations orartificial mutations. Said mutations of course should allow detectionwith a specific binding member of HCoV-NL63, not necessarily with all ofthe specific binding members). Said mutations should allow the detectionof the variants using common detection methods such as antibodyinteraction, amplification and/or hybridization.

Considering that specific binding members are important molecules forinstance for diagnostic purposes, the invention further provides the useof a virus of the invention or functional part, derivative and/oranalogue thereof, for detecting a molecule capable of specificallybinding said virus in a sample. Further provided is the use of a nucleicacid and/or amino acid sequence of a virus or functional part,derivative or analogue as defined by the invention, or detecting amolecule capable of specifically binding said virus or functional part,derivative and/or analogue in a sample. Preferably said nucleic acidand/or amino acid sequence comprises a sequence as depicted in table 3or Table 3 or a functional part, derivative or analogue thereof.Preferably said part is at least 30 nucleotides and/or amino acids longwherein said part preferably comprises more than 95% sequence identity,preferably more than 99%. In a preferred aspect said specific bindingmember comprises a specific ligand and/or antibody of said virus.

Further provided is a primer and/or probe according to the invention, aspecific binding member of the invention, and/or a nucleic acid of avirus or functional part, derivative or analogue according to theinvention, for detecting and/or identifying a HCoV-NL63 coronavirus orpart thereof in a sample. Preferably, said nucleic acid comprises asequence as depicted in table 3.

HCoV-NL63 virus may be used to generate an immune response in a subject.This can be useful for instance in vaccination strategies. Thus theinvention further HCoV-NL63 provides HCoV-NL63 virus or functional part,derivative or analogue thereof for use as a vaccine or medicament. Themedicament use is typically when the subject is already infected withthe virus and the immunogen is used to augment the immune responseagainst the virus. The invention further provides a specific bindingmember of the invention for use as a vaccine or medicament This use isparticularly favorable for when the specific binding member comprises aproteinaceous molecule, preferably an antibody or functional part,derivative and/or analogue thereof. Such an antibody can provide passiveimmunity but may also have active components such as proteases attachedto it. The medicament use may again be the case wherein a subjectinfected with an HCoV-NL63 virus is treated with the specific bindingmember.

Vaccines may be generated in a variety of ways. One way is to culturethe HCoV-NL63 virus for example on the mentioned monkey cell line(s) andto use inactivated virus harvested from the culture. Alternatively,attenuated virus may be used either inactivated or as a live vaccine.Methods for the generation of coronavirus vaccines may be adapted toproduce vaccines for the HCoV-NL63 of the invention. The invention thusfurther provides the use of an HCoV-NL63 virus or functional part,derivative or analogue thereof for the preparation of a vaccine againsta coronaviral genus related disease. The invention further provides theuse of a specific binding member of the invention for the preparation ofa vaccine or medicament against a coronaviral genus related disease.Further provided is the use of an HCoV-NL63 virus or functional part,derivative or analogue thereof, a specific binding member of theinvention, a nucleic acid of the invention or a primer and/or probe ofthe invention for diagnosis of a coronaviral genus related disease.Preferably said coronaviral genus related disease comprises a HCoV-NL63coronavirus related disease.

Further provided is a vaccine comprising an HCoV-NL63 virus orfunctional part, derivative or analogue thereof and/or a specificbinding member of the invention. Also provided is a medicamentcomprising an HCoV-NL63virus or functional part, derivative or analoguethereof and/or a specific binding member of the invention. Preferablysaid vaccine or medicament is used for at least in part preventingand/or treating a HCoV-NL63 related disease.

An important use of the present invention is the generation of adiagnostic tool for determining whether a subject is suffering from anHCoV-NL63 virus infection or has been exposed to an HCoV-NL63 virusinfection. Many diff rent diagnostic applications can be envisioned.They typically contain an identifying component allowing the typing ofthe virus that is or was present in the subject. One diagnostic tool forHCoV-NL63 makes use of the particular proliferation characteristics ofthe virus in various cell lines. It replicates in the mentionedpreferred monkey cell lines but does not replicate in Vero-cells. Thisproperty can be used to discriminate HCoV-NL63 from other knowncoronaviruses. Thus in one aspect the invention provides a diagnostickit comprising at least one of the preferred monkey cell lines,preferably the tertiary monkey kidney cells (tMK; Cynomolgus monkey orthe monkey cell line LLC-MK2.

Many modern diagnostic kits comprise a specific binding member (todetect the virus or virus infected cells) and/or an HCoV-NL63 virus or afunctional part, derivative and/or analogue thereof and/or ammo acid ofthe invention or a functional part, derivative and/or analogue thereof(for detecting antibodies in blood components of the diagnosed subject).Many other current diagnostic kits rely on identification of HCoV-NL63virus specific nucleic acid in a sample. There are various ways in whichsuch an assay may be implemented one is a method for detecting anHCoV-NL63 virus or functional part, derivative or analogue thereof in asample, comprising hybridizing and/or amplifying a nucleic acid of saidvirus or functional part, derivative or analogue with a primer and/orprobe according to the invention and detecting hybridized and/oramplified product. The invention thus also provides a diagnostic kitcomprising an HCoV-NL63 virus or functional part, derivative or analoguethereof, a specific binding member according to the invention and/or aprimer/probe according to the invention.

Further provided is a method for treating an individual suffering from,or at risk of suffering from, a HCoV-NL63 related disease, comprisingadministering to said individual a vaccine or medicament according tothe invention. Also provided is a method for determining whether anindividual suffers from a HCoV-NL63 related disease, comprisingobtaining a sample from said individual and detecting a HCoV-NL63 virusor functional part, derivative or analogue thereof in said sample with amethod and/or diagnostic kit of the invention.

Further provided is an isolated or recombinant nucleic acid encoding avirus or functional part, derivative and/or analogue according to theinvention and a nucleic acid according to the invention, comprising atleast a functional part of a sequence as depicted in Table 3. Furtherprovided is an amino acid sequence encoded by a nucleic acid accordingto the invention, and an amino acid sequence according to the invention,comprising at least a functional part of a sequence as depicted in Table3. Further provided is a proteinaceous molecule capable of specificallybinding HCoV-NL63, obtainable by a method according to the inventionand, the use of such a proteinaceous molecule in a vaccine or adiagnostic method for the detection of HCoV-NL63.

EXAMPLES Example 1

cDNA-AFLP for Virus Discovery

We modified the cDNA-AFLP technique such that it can amplify viralsequences from blood-plasma/serum samples or from CPE-positive culturesupernatants (FIG. 1). In the adjusted method the mRNA isolation stepprior to amplification is replaced by a treatment to purify viralnucleic acid. Of importance to the purification is a centrifugation stepto remove residual cells and mitochondria. In addition, a single DNAsetreatment is sufficient to get rid of interfering chromosomal DNA andmitochondrial DNA from broken down cells and finally, by choosingfrequent cutting restriction enzymes, the method is fine-tuned such thatthe majority of viruses will be amplified. With this so-called VirusDiscovery cDNA-AFLP (VIDISCA) we were able to amplify viral nucleicacids from EDTA-plasma of a person with hepatitis B virus infection anda person with an acute Parvo B19 infection (results not shown). Thetechnique can also detect HIV-1 in a positive culture supernatantdemonstrating its capacity to identify both RNA and DNA viruses (resultsnot shown).

To eliminate residual cells, 110 μl of virus culture supernatant wasspun down for 10 min at maximum speed in an Eppendorf microcentrifuge(13500 rpm). One hundred μl was transferred to a fresh tube and DNAsetreated for 45 minutes at 37° C. using 15 μl of DNAse buffer and 20Units of DNAse I (Ambion). The DNAse treatment was included to get ridof chromosomal DNA from broken down cells. After this 900 μl of L6 lysisbuffer and 40 μl of silica suspension was added and nucleic acids wereextracted as described by Boom⁴. The viral nucleic acids were eluted in40 μl H₂O. With 20 μl eluate the reverse transcription was performedusing 2.5 μg random hexamers (Amersham Bioscience), 200 U MMLV-RT(InVitrogen) in a buffer containing 10 mM Tris-HCl pH 8.3, 50 mM KCl,0.1% Triton X-100, 4.8 mM MgCl2, and 0.4 mM of each dNTP. The sample wasincubated at 37° C. for 90 minutes. Subsequently the second strand DNAsynthesis was performed using 26 U Sequenase II (Amersham Bioscience),7.5 U RNAse H (Amersham Bioscience) in 0.25 mM dNTPs each, 17.5 mM MgCl2and 35 mM Tris-HCl pH 7.5. After the incubation at 37° C. for 90 minutesa phenol/chloroform extraction was performed followed by an ethanolprecipitation. The pellet was dissolved in 30 μl of H₂O. The cDNA-AFLPwas performed essentially as described by Bachem¹ with somemodifications. The dsDNA was digested with the HinP I and MseIrestriction enzymes (New England Biolabs) according to the manufacturersprotocol. After the digestion, MseI adaptor and HinP I adaptor (seebelow) are added together with 5U ligase enzyme (InVitrogen) and ligasebuffer, followed by an additional incubation of 2 hrs at 37° C. The MseIadaptor and HinP I adaptor were prepared previously by mixing a topstrand oligo for the MSE and the HinPI adaptors (Table 1) with a bottomstrand oligo for the MSE adaptor and for the HinPI adaptor, incubate at65° C. followed by cooling down to room temperature in the presence of a1:40 dilution of ligase buffer.

The first PCR was performed with 10 μl of ligation mixture as input, 2.5U of AmpliTaq polymerase (Perkin-Elmer), 100 ng of HinPI standard primerand 100 ng of MseI standard primer. The PCR reaction was performedaccording to the profile 5 min 95C; 20 cycles of: 1 min 95° C.−1 min 55°C.−2 min 72° C.; 10 min 72° C. Five μl of first PCR product was used asinput in the second “selective” amplification step containing 100 ng ofHinPI-N primer and 100 ng MseI-N (sequence of the standard primersextended with one nucleotide) and 2 U AmpliTaq polymerase. The selectivePCRs were amplified according to the profile of the “touch down PCR”: 10cycles of 60 sec 94° C.−30 sec 65° C.−1 min 72° C. over which theannealing temperature was reduced from 65° C. with 1° C. with eachcycle, followed by 23 cycles: 30 sec 94° C.−30 sec 56° C.−1 min 72° C.Finally the sample was incubated for 10 min at 72° C. The PCR productswere evaluated on 4% Metaphor® gels (Cambrex, Rockland, USA). If thebands on the gel were very faint the PCR products were concentrated byvacuum drying using 60 μl of the PCR product. The PCR fragments ofinterest were cut out of gel and DNA was eluted from the gel using theQiagen gel purification kit according to the manufacturer's protocol.The PCR products were cloned using pCR® 2.1-TOPO plasmid (InVitrogen)and chemically competent One Shot E. coli (InVitrogen). A PCR on thecolony was performed and this PCR product was input for sequencing theinsert using Big Dye terminator chemistry (Applied Biosystems). Thereverse transcription step was excluded, only HinP I digestion andadaptor ligation was performed, the first PCR was performed with 35cycles instead of 20 and those first PCR fragments were visualized onagarose gel electrophoresis.

DNA Sequencing and Analysis

Coronavirus-PCR product containing plasmids were sequenced with theBigDyeTM Terminator Cycle Sequencing Kit (Applied Biosystems, FosterCity, Calif.), using the −21 M13RP and T7 primers. Electrophoresis ofsequencing reaction mixtures was performed with an Applied Biosystems377 automated sequencer, following the manufacturer's protocols. TheSequence Navigator (version 1.01) and Auto Assembler (version 2.1)software packages (ABI, Calif., USA) were used to analyze all sequencingdata. Sequences were compared to all sequences in the Genbank databaseusing the BLAST tool of the NCBI web page:http://www.ncbi.nlm.nih.gov/blast. For phylogenetic analysis thesequences were aligned using the ClustalX software package³⁴ with thefollowing settings: Gap opening penalties: 10.00; Gap extension penalty0.20, Delay divergent sequences switch at 30% and transition weight0.59. Phylogenetic analysis was carried out using the neighbor-joiningmethod of the MEGA program (9). The nucleotide distance matrix wasgenerated either by Kimura's 2-parameter estimation or by the p-distanceestimation (5), Bootstrap resampling (500 replications) was employed toplace approximate confidence limits on individual branches.

Determining the Nucleotide Sequence of the Complete HCoV-NL63 Genome

Using a combination of specific primers, located in the alreadysequenced domains of the HCoV-NL63 genome, and the proprietaryPALM-method (WO 0151661) we are in the process of cloning anddetermining the full-length genomic sequence for this new coronavirus.Using a combination of 5′-oligonucleotides located in the analyzed partof the HCoV-NL63 genome and a 3′ tagged random primer (JZH2R) additionalfragments were amplified using a nested RT-PCR protocol similar to theone mentioned previously.

Isolation of SZ 163

In January 2008 a 7-month-old child appeared in hospital with coryza,conjunctivitis and fever. Chest radiography showed typical features ofbronchiolitis and four days after the onset of disease a nasopharyngealaspirate specimen was collected (sample nr: HCoV-NL63). All routinelyused tests on this sample for adenovirus, respiratory syncytial virus(RSV), influenza A and B, parainfluenza 1, 2 and 3, rhinovirus,HCoV-229E and HCoV-OC43 were negative. The clinical sample wassubsequently inoculated onto a variety of cells including humanfibroblast lung (HFL) cells, tertiary monkey kidney cells (tMK;Cynomolgus) and R-HeLa cells. A CPE was detected exclusively on tMKcells and first noted at eight days post-inoculation. The CPE wasdiffuse with a refractive appearance in the affected cells followed bycell detachment after 7 days. More pronounced CPE was observed uponpassage onto LLC-MK2 cells. Besides overall cell rounding, moderate cellenlargement was observed. Additional subculturing on human endotheliallung cells, HFL, Rhabdomyosarcoma cells and Vero cells remained negativefor CPE. Immunofluorescent assays to detect influenzavirus A and B, RSV,adenoviruses or parainfluenza virus types 1, 2 or 3 in the cultureremained negative The culture supernatant of infected LLC-MK2 cells wassubsequently analyzed by VIDISCA. As control we used the supernatant ofuninfected LLC-MK2 cells. After the second PCR amplification step,several DNA fragments were present in the test sample but not in thecontrol. These fragments were cloned and sequenced. A Blast search inGenBank revealed that 8 of 16 fragments had sequence similarity to thefamily of corona viruses with the highest homology the human coronavirus 229E Cables 4 and 5).

Phylogenetic analysis of a 270 nt fragment of the replicase 1B regionindicated that we identified a distinct new member of the coronavirusgroup 1. With the VIDISCA technique, 8 HCOV-163-specific fragments,named 163-2, 163-4, 163-9, 163-10, 163-11, 163-14, 163-15 and 163-18were isolated, cloned, sequenced and aligned with the relevant sequencesfrom GenBank. The Genbank accession number of the used sequences are:MHV (mouse hepatitis virus): AF201929; HCoV-229E: AF304460; PEDV(porcine epidemic diarrhea virus): AF353511; TGEV (transmissiblegastroenteritis virus). AJ271965; SARS-CoV: AY278554; IBV (avianinfectious bronchitis virus): NC_(—)001451; BCoV (bovine coronavirus):NC_(—)008045; FCoV (feline coronavirus): Y13921 and X80799; CCoV (caninecoronavirus): AB105373 and A22732; PRCoV (porcine respiratorycoronavirus): M94097; FIPV (feline infectious peritonitis virus):D32044. Position of the HCoV-NL63 fragments compared to HCoV-229E(AF304460): Replicase 1AB gene: 15156-15361, 16049-16182, 16190-16315,18444-18550, Spike gene: 22124-22266; Nucleocapsid gene: 25667-25882 and25887-25957; 3′UTR: 27052-27123. Branch lengths indicate the number ofsubstitutions per sequence. From the most closely related speciessequence identity scores were calculated (Tables 5 and 6).

Also the deduced amino acid sequence were aligned to the correspondingdomains in the open reading frames of related corona (-like) viruses(Table 6).

The human corona viruses account for 10 to 30% of the common colds inman⁷, and it is not unusual to find a coronavirus in a child with arespiratory illness. However, it is striking that the virus HCoV-NL63was harvested from LLC-MK cells. Human Corona virus 229E and OC-43 areknown for there inability to replicate on monkey kidney cells.Intriguingly, the newly identified human corona virus that isresponsible for SARS is also able to replicate in monkey kidney cells³⁰.

Propagation of HCoV-NL63 in Cell Culture

A nasopharyngeal aspirate was collected 4 days after the onset ofsymptoms. The specimen was tested for the presence of adenovirus, RSV,influenza A, influenza B, and parainfluenza type 1, 2 an 3 using theVirus Respiratory Kit (Bartels: Trinity Biotech plc, Wicklow Ireland).In addition, PCR diagnosis for rhinoviruses, meta-pneumovirus andHCoV-OC43 and HCoV-229E were performed^(2, 10). The originalnasopharyngeal aspirate was subsequently inoculated onto a variety ofcell cultures including HFL cells, tMK cells and R-HeLa cells. The tubeswere kept in a roller drum at 34° C. and observed every 3 to 4 days.Maintenance medium was replenished every 3 to 4 days. Two differenttypes of medium were implemented: Optimem 1 (Gibco) without bovine fetalserum was used for the tMK cells and MEM Hanks'/Earle's medium (Gibco)with 3% bovine fetal serum was used for the remaining cell types. On thevirus culture direct staining was performed with pools offluorescent-labeled mouse antibodies against influenzavirus A and B, RSVand adenoviruses (Imagen, DAKO). Indirect staining was performed forparainfluenza virus types 1, 2 or 3 with mouse antibodies (Chemicon,Brunschwig, Amsterdam Netherlands) and subsequent staining with labeledrabbit anti-mouse antibodies (Imagen, DAKO).

Method to Detect HCoV-NL63 in Nasopharyngeal Swabs

For the diagnostic RT-PCR, nucleic acids were extracted by the Boommethod⁴ 4 from 50 μl virus supernatant or 50 μl suspended nasopharyngealswab. The reverse transcription was performed as described above withthe exception that 10 ng of reverse transcription primer repSZ-RT (Table7) was used. The entire RT mixture was added to the first PCR mixturecontaining 100 ng of primer repSZ-1 and 100 ng of primer repSZ-3. ThePCR reaction was performed according to the profile 5 min 95° C.; 20cycles of: 1 min 95° C.−1 min 55° C.−2 min 72° C.; 10 min 72 ° C. Anested PCR was started using 5 μl of the first PCR with 100 ng of primerrepSZ-2 and 100 ng of primer repSZ-4. Twenty-five PCR cycles wereperformed of the same profile as the first PCR.

Ten μl of the first and 10 μl of the nested PCR was analyzed by agarosegel electrophoresis (FIG. 2). Cloning and sequencing of the fragmentswas performed essentially as described above.

Method of Raising Polyclonal Antibodies

Appropriate domains within the HCoV-NL63 surface proteins (e.g.S-glycoprotein or HE-glycoprotein) can be selected and amplified withsuitable oligonucleotides and RT-PCR. The corresponding purified viralantigens can be obtained by expression in a suitable host (e.g. Yarrowialipolytica as previously described³⁸). Female NZW rabbits (approx 4 kg)are primed with 0.5 to 5.0 mg of viral protein antigen preparation. Theantigen is suspended in 0.5 ml. of phosphate buffered saline (pH 7.3)and emulsified in an equal volume of complete Freund's adjuvant (CFA).Freund's Adjuvant is a well-established adjuvant system that isappropriate for use in these experiments where small amounts of antigenare used, and where immunogenicity of the antigen (although likely) isunknown. Published guidelines for use will be followed, includinglimiting injection to 0.1 ml at each site, using CFA only for initialimmunization dose. This antigen preparation (1 ml total volume) isinjected subdermally in the loose skin on the backside of the rabbit'sneck. This injection route is immunologically effective and minimizesthe possibility of local inflammation associated with unilateral orbilateral flank injection (such ensuing flank inflammation can impairanimal mobility). After resting for 3 weeks, one ml of blood will beremoved from the ear artery for a test bleed. Antibodies will be boostedif titers of the desirable antibodies are judged to be too low. Rabbitswith adequate antibody levels will be boosted subdermally 1.0 mg ofantigen contained in CFA. Boosted animals will be bled after two weeks;i.e., 15 ml of blood will be taken from the ear artery using a heat lampto dilate the blood vessel. The rabbit will be placed in a commercialrestraint, tranquillized with xylazine not more than seven times intotal after which the rabbit will be exsanguinated by cardiac puncturefollowing anesthesia using xylazine/ketamine.

Method for Vaccine Production

For the production of a subunit vaccine the S-glycoprotein perhapscombined with the HE, M and N proteins, could be expressed in a suitableeukaryotic host (e.g. Y. lipolytica or LLC-MK2 cells) and purified usingpreferentially two small affinity tags (e.g. His-tag or the StrepIItag). After appropriate purification, the resulting viral proteins canbe used as a subunit vaccine.

Alternatively the HCoV-NL63 virus can be propagated in a suitable cellline as described above and subsequently treated as described by Wu¹¹.Briefly the virus is precipitated from culture medium with 20%polyethylene glycol 6000 and purified by ultracentrifugation at 80.000×gfor 4 hours through a discontinuous 40-65% sucrose gradient followed bya linear 5 to 40% CsCl gradient for 4 hours at 120.000×g. The resultingvirus preparation can be inactivated by beating for 30 minutes at 65° C.as described by Blondel³.

Analysis of S Glycoprotein or Any of the HCOV-NL63 Viral ProteinsBinding to an Immobilized Ligand (e.g. Antibody) in an Optical Biosensor

Binding reactions were carried out in an IAsys two-channel resonantmirror biosensor at 20° C. (Affinity Sensors, Saxon Hill, Cambridge,United Kingdom) with minor modifications. Planar biotin surfaces, withwhich a signal of 600 arc s corresponds to 1 ng of bound protein/mm2,were derivatized with streptavidin according to the manufacturer'sinstructions. Controls showed that the viral proteins did not bind tostreptavidin-derivatized biotin surfaces (result not shown).Biotinylated antibody was immobilized on planar streptavidin-derivatizedsurfaces, which were then washed with PBS. The distribution of theimmobilized ligand and of the bound S-glycoprotein on the surface of thebiosensor cuvette was inspected by the resonance scan, which showed thatat all times these molecules were distributed uniformly on the sensorsurface and therefore were not micro-aggregated. Binding assays wereconducted in a final volume of 30 μl of PBS at 20±0.1° C. The ligate wasadded at a known concentration in 1 μl to 5 μl of PBS to the cuvette togive a final concentration of S-glycoprotein ranging from 14 to 70 nM.To remove residual bound ligate after the dissociation phase, and thusregenerate the immobilized ligand, the cuvette was washed three timeswith 50 μl of 2 M NaCl-10 mM Na2HPO4, pH 7.2, and three times with 50 μlof 20 mM HCl. Data were pooled from experiments carried out withdifferent amounts of immobilized antibody (0.2, 0.6, and 1.2 ng/mm2).For the calculation of k_(on), low concentrations of ligate(S-glycoprotein) were used, whereas for the measurement of k_(off),higher concentrations of ligate were employed (1 μM) to avoid anyrebinding artefacts. The binding parameters k_(on) and k_(off) werecalculated from the association and dissociation phases of the bindingreactions, respectively, using the nonlinear curve-fitting FastFitsoftware (Affinity Sensors) provided with the instrument. Thedissociation constant (K_(d)) was calculated from the association anddissociation rate constants and from the extent of binding observed nearequilibrium.

Example 2 Methods

Virus Isolation

The child, who was living in Amsterdam, was admitted to the hospitalwith complaints of coryza and conjunctivitis since 3 days. At admissionshe had shortness of breath and refused to drink. The patient'stemperature was 89° C., the respiratory rate was 50 breaths/min withoxygen saturation of 96% and her pulse was 177 beats/min. Uponauscultation bilateral prolonged expirium and end-expiratory wheezingwas found. A chest radiograph showed the typical features ofbronchiolitis. The child was treated with salbutamol and ipratropium atthe first day, followed by the use of salbutamol only for 5 days. Thechild was seen daily at the out patient clinic and the symptomsgradually decreased. A nasopharyngeal aspirate was collected 5 daysafter the onset of symptoms. The specimen was tested for the presence ofRSV, adenovirus, influenza A and B virus, and parainfluenza virus type1, 2 and 3 using the Virus Respiratory Kit (Bartels: Trinity Biotechplc, Wicklow Ireland). In addition, PCR tests for rhinoviruses,enterovirus, meta-pneumovirus and HCoV-OC43 and HCoV-229E were performed(2, 10). The original nasopharyngeal aspirate was inoculated onto avariety of cells. The cultures were kept in a roller drum at 34° C. andobserved every 3 to 4 days. Maintenance medium was replenished every 3to 4 days. Two different types of medium were implemented: Optimem 1(InVitrogen, Breda, The Netherlands) without bovine fetal serum was usedfor the tMK cells and MEM Hanks'/Earle's medium (InVitrogen, Breda, TheNetherlands) with 3% bovine fetal serum was used for the remaining celltypes. Cell cultures that were infected with the aspirate specimen werestained for the presence of respiratory viruses after one week ofincubation. Direct staining was performed with pools offluorescent-labeled mouse antibodies against RSV and influenza A and Bvirus (Imagen, DakoCytomation Ltd, Cambridge, UK). Indirect staining wasperformed for adenoviruses and parainfluenza virus type 1, 2 or 3 withmouse antibodies (Chemicon International, Temecula, Calif.) andsubsequent staining with FITC-labeled rabbit anti-mouse antibodies(Imagen, DakoCytomation Ltd, Cambridge, UK.

VIDISCA method

To remove residual cells and mitochondria, 110 μl of virus culturesupernatant was spun down for 10 min at maximum speed in an eppendorfmicrocentrifuge (13500 rpm). To remove chromosomal DNA and mitochondrialDNA from the lysed cells, 100 μl was transferred to a fresh tube andtreated with DNAse I for 45 min at 37° C. (Ambion, Huntingdon, UK).Nucleic acids were extracted as described by Boom et al. (4). A reversetranscription reaction was performed with random hexamer primers(Amersham Bioscience, Roosendaal, The Netherlands) and MMLV-RT(InVitrogen, Breda The Netherlands) while second strand DNA synthesiswas carried out with Sequenase II (Amersham Bioscience, Roosendaal, TheNetherlands). A phenol/chloroform extraction was followed by an ethanolprecipitation. The cDNA-AFLP was performed essentially as described byBachem et al (1) with some modifications. The dsDNA was digested withthe HinP I and Mse I restriction enzymes (New England Biolabs, Beverly,Massachusetts). Mse I- and HinP I-anchors (see below) were subsequentlyadded with 5U ligase enzyme (InVitrogen, Breda, The Netherlands) in thesupplied ligase buffer for 2hrs at 37° C. The Mse I- and HinP I-anchorswere prepared by mixing a top strand oligo (5′-CTCGTAGACTGCGTACC-3′(SEQ. ID. NO: 3) for the Mse I anchor and 5′-GACGATGAGTCCTGAC-3′ (SEQ.ID. NO: 4) for the HinP I anchor) with a bottom strand oligo(5′-TAGGTACGCAGTC-3′ (SEQ. ID. NO: 5) for the Mse I anchor and5′-CGGTCAGGACTCAT-3′ (SEQ. ID. NO: 6) for the HinP I anchor) in a 1:40dilution of ligase buffer. A 20 cycle PCR was performed with 10 μl ofthe ligation mixture, 100 ng HinP I standard primer(5′-GACGATGAGTCCTGACCGC-3′ (SEQ. ID. NO: 7)) and 100 ng Mse I standardprimer (5′-CTCGTAGACTGCGTACCTAA-3′ (SEQ. ID. NO:8)). Five μl of this PCRproduct was used as input in the second “selective” amplification stepwith 100 ng HinPI-N primer and 100 ng MseI-N (the “N” denotes that thestandard primers are extended with one nucleotide: G, A, T or C). Theselective rounds of amplification were done with a “touch down PCR”: 10cycles of [60 sec 94° C-30 sec 65° C-1 min 72° C.] and the annealingtemperature was reduced with 1 ° C. each cycle, followed by 23 cycles:[30 sec 94° C-30 sec 56 ° C-1 min 72° C.] and 1 cycle 10 min 72° C. ThePCR products were analyzed on 4% Metaphor® agarose gels (Cambrex,Rockland, Maine) and the fragments of interest were cloned and sequencedusing BigDye terminator reagents. Electrophoresis and data collectionwas performed on an ABI 377 instrument.

cDNA Library Construction and Full Genome Sequencing

The cDNA library was produced as described by Marra et al¹⁷, with minormodifications. During reverse transcription only random hexamer primerswere used and no oligo-dT primer, and the amplified cDNA was cloned intoPCR2.1-TOPO TA clong vector. Colonies were picked and suspended in BHImedia. The E. coli suspension was used as input in a PCR amplificationusing T7 and M13 RP for amplification. The PCR products weresubsequently sequenced with the same primers that were used in thePCR-amplification and the BigDye terminator reagent. Electrophoresis anddata collection was performed on an ABI 377 instrument. Sequences wereassembled using the AutoAssembler DNA sequence Assembly software version2.0.

Diagnostic RT-PCR

From 492 persons a total of 600 respiratory samples collected betweenDecember 2002 and August 2002. The kind of material ranged fromoral/nasopharyngeal aspirate, throat swabs, bronchioalveolary lavagesand sputum. The samples were collected for routine virus diagnosticscreening of persons suffering from upper and lower respiratory tractdisease. One hundred μl of the sample was used in a Boom extraction (4).The reverse transcription was performed with MMLV-RT (InVitrogen) using10 ng or reverse transcription primer (repSZ-RT: 5′- CCACTATAAC-3′ (SEQ.ID. NO: 9)). The entire RT mixture was added to the first PCR mixturecontaining 100 ng of primer repSZ-1 (5′-GTGATGCATATGCTAATTTG-3′ (SEQ.ID. NO: 10)) and 100 ng of primer repSZ-3 (5′-CTCTTGCAGGTATAATCCTA-3′(SEQ. ID. NO: 11)). The PCR reaction was performed according to theprofile 5min 95° C.; 20 cycles of: 1min 95° C.-1min 55° C.-2min 72° C.;10 min 72 ° C. A nested PCR was started using 5 μl of the first PCR with100 ng of primer repSZ-2 (5′-TTGGTAAACAAAAGATAACT-3′ (SEQ. ID. NO: 12))and 100 ng of primer repSZ-4 (5′-TCAATGCTATAAACAGTCAT-3′ (SEQ. ID. NO:13)). Twenty-five PCR cycles were performed of the same profile as thefirst PCR. Ten μl of the PCR products was analyzed by agarose gelelectrophoresis. All positive samples were sequenced to confirm thepresence of HCoV-NL63 in the sample.

Sequence Analysis

Sequences were compared to all sequences in the Genbank database usingthe BLAST tool of the NCBI web page: http://www.ncbi.nlm.nih.gov/blast.For phylogenetic analysis the sequences were aligned using the ClustalXsoftware package with the following settings: Gap opening penalties:10.00; Gap extension penalty 0.20; Delay divergent sequences switch at30% and transition weight 0.5 (9). Phylogenetic analysis was carried outusing the neighbor-joining method of the MEGA program (5) using theinformation of all fragments within one gene. The nucleotide distancematrix was generated either by Kimura's 2 parameter estimation or by thep-distance estimation (6). Bootstrap resampling (500 replicates) wasemployed to place approximate confidence limits on individual branches.

Results

Virus Isolation From a Child With Acute Respiratory Disease

In January 2003 a 7-month-old child appeared in the hospital withcoryza, conjunctivitis and fever. Chest radiography showed typicalfeatures of bronchiolitis and a nasopharyngeal aspirate specimen wascollected five days after the onset of disease (sample NL63). Diagnostictests for respiratory syncytial virus (RSV), adenovirus, influenza A andB virus, parainfluenza virus type 1, 2 and 3, rhinovirus, enterovirus,HCoV-229E and HCoV-OC43 remained negative. The clinical sample wassubsequently inoculated onto human fetal lung fibroblasts (HFL),tertiary monkey kidney cells (tMK; Cynomolgus monkey) and HeLa cells.CPE was detected exclusively on tMK cells and first noted at eight dayspost-inoculation. The CPE was diffuse with a refractive appearance inthe affected cells followed by cell detachment after 7 days. Morepronounced CPE was observed upon passage onto the monkey kidney cellline LLC-MK2 with overall cell rounding and moderate cell enlargement(FIG. 1). Additional subcultures on HFL, rhabdomyosarcoma cells and Verocells remained negative for CPE. Immunofluorescent assays to detect RSV,adenovirus, influenza A and B virus, or parainfluenza virus type 1, 2and 3 in the culture remained negative. Acid lability and chloroformsensitivity tests demonstrated that the virus is most likely envelopedand not a member of the picornavirus group²⁴.

Virus Discovery by the VIDISCA Method

Identification of unknown pathogens by molecular biology toolsencounters the problem that the target sequence is not known and thatgenome specific PCR-primers cannot be designed. To overcome this problemwe developed the VIDISCA method that is based on the cDNA-AFLPtechnique⁴. The advantage of VIDISCA is that prior knowledge of thesequence is not required as the presence of restriction enzyme sites issufficient to guarantee amplification. The input sample can be eitherblood plasma/serum or culture supernatant. Whereas cDNA-AFLP starts withisolated mRNA, the VIDISCA technique begins with a treatment toselectively enrich for viral nucleic acid, which includes acentrifugation step to remove residual cells and mitochondria. Inaddition, a DNAse treatment is used to remove interfering chromosomalDNA and mitochondrial DNA from degraded cells, whereas viral nucleicacid is protected within the viral particle. Finally, by choosingfrequently cutting restriction enzymes, the method is fine-tuned suchthat most viruses will be amplified. Using VIDISCA we were able toamplify viral nucleic acids from EDTA-plasma of a person with hepatitisB virus infection and a person with an acute parvovirus B19 infection.The technique can also detect HIV-1 in cell culture, demonstrating itscapacity to identify both RNA and DNA viruses.

The supernatant of the CPE-positive culture NL63 was analyzed byVIDISCA. We used the supernatant of uninfected cells as a control. Afterthe second PCR amplification step, unique and prominent DNA fragmentswere present in the test sample but not in the control. These fragmentswere cloned and sequenced. Twelve out of 16 fragments showed sequencesimilarity to members of the family of coronaviruses, but significantsequence divergence was apparent in all fragments. These resultsindicate that we identified a novel coronavirus (HCoV-NL63).

Detection of HCoV-NL63 in Patient Specimens

To demonstrate that HCoV-NL63 originated from the nasopharyngealaspirate of the child, we designed a diagnostic RT-PCR that specificallydetects HCoV-NL63. This test, based on unique sequences within the 1bgene, confirmed the presence of HCoV-NL63 in the clinical sample. Thesequence of this PCR product was identical to that of the virusidentified upon in vitro passage in LLC-MK2 cells (results not shown).

Having confirmed that the cultured coronavirus originated from thechild, the question remains whether this is an isolated clinical case orwhether HCoV-NL63 is circulating in humans. To address this question, weexamined respiratory specimens of hospitalized persons and individualsvisiting the outpatient clinic between December 2002 and August 2008 forthe presence of HCoV-NL63. We identified 7 additional persons thatcarried HCoV-NL63. Sequence analysis of the PCR products indicated thepresence of a few characteristic (and reproducible) point mutations inseveral samples, suggesting that several subgroups of NL63 mayco-circulate. At least 5 of the HCoV-NL63-positive individuals sufferedfrom a respiratory tract illness, the clinical data of 2 persons werenot available. Including the index case, five patients were childrenless than 1 year old and 3 patients were adults. Two adults are likelyto be immuno-suppressed, as one of them is a bone marrow transplantrecipient, and the other is an HIV positive patient suffering from AIDSwith very low CD4 cell counts. No clinical data of the third adult wasavailable. Only 1 patient had a co-infection with RSV (nr 72), and theHIV-infected patient (nr 466) carried Pneumocystis carinii. No otherrespiratory agent was found in the other HCoV-NL63-positive patients,suggesting that the respiratory symptoms were caused by HCoV-NL63. AllHCoV-NL63 positive samples were collected during the last winter season,with a detection frequency of 7% in January 2003. None of the 306samples collected in the spring and summer of 2003 contained the virus(P<0.01, 2-tailed t-test).

Complete Genome Analysis of RCoV-NL63

The genomes of coronaviruses have a characteristic, genome organization.The 5′ half contains the large 1a and 1b genes, encoding thenon-structural polyproteins, followed by the genes coding for fourstructural proteins: spike (S), membrane (M), envelope (E) and thenucleocapsid (N) protein. Additional non-structural proteins are encodedeither between 1b and the S gene, between the S and E gene, between theM and N gene or downstream of the N gene.

To determine whether the HCoV-NL63 genome organization shares thesecharacteristics, we constructed a cDNA library with a purified virusstock as input material. A total of 475 genome fragments were analyzed,with an average coverage of 7 sequences per nucleotide. Specific PCRswere designed to fill in gaps and to sequence regions with low qualitysequence data. Combined with 5′RACE (Rapid Amplification of cDNA Ends)and 3′RACE experiments the complete HCoV-NL63 genome sequence wasresolved.

The genome of HCoV-NL63 is a 27,553-nucleotide RNA with a poly A tail.With a G-C content of 34% it has the lowest G-C content among thecoronaviridae, which range from 37%-42%²⁵. ZCurve software was used toidentify ORFs²⁶ and the genome configuration is portrayed using thesimilarity with known coronaviruses (FIG. 6). The 1a and 1b genes encodethe RNA polymerase and proteases that are essential for virusreplication. A potential pseudoknot structure is present at position12489, which may provide the −1 frameshift signal to translate the 1bpolyprotein. Genes predicted to encode the S, E, M and N proteins arefound in the 3′ part of the genome. Short untranslated regions (UTRs) of286 and 287 nucleotides are present at the 5′ and 3+ termini,respectively. The hemagglutinin-esterase gene, which is present in somegroup 2 and group 3 coronaviruses, was not present. ORF 3 between the Sand E gene probably encodes a single accessory non-structural protein.

The 1a and 1ab polyproteins are translated from the genomic RNA, but theremaining viral proteins are translated from subgenomic mRNAs (sg mRNA),each with a common 5′ end derived from the 5′ part of the genome (the 5′leader sequence) and 3′ coterminal parts. The sg mRNA are made bydiscontinuous transcription during negative strand synthesis²⁷.Discontinuous transcription requires base-pairing between cis-actingtranscription regulatory sequences (TRSs), one located near the 5′ partof the genome (the leader TRS) and others located upstream of therespective ORFs (the body TRSs)²⁸. The cDNA bank that we used forsequencing contained copies of sg mRNA of the N protein, thus providingthe opportunity to exactly map the leader sequence that is fused to allsg mRNAs. A leader of 72 nucleotides was identified at the 5′ UTR. Theleader TRS (5′-UCUCAACUAAAC-3′ (SEQ. ID. NO: 14)) showed11/12-nucleotide similarity with the body TRS upstream of the N gene. Aputative TRS was also identified upstream of the S, ORF 3, E and M gene

The sequence of HCoV-NL63 was aligned with the complete genomes of othercoronaviruses. The percentage nucleotide identity was determined foreach gene. For all genes except the M gene, the percentage identity wasthe highest with HCoV-229E. To confirm that HCoV-NL63 is a new member ofthe group 1 coronaviruses, phylogenetic analysis was performed using thenucleotide sequence of the 1A, 1B, S, M and N gene. For each geneanalyzed, HCoV-NL63 clustered with the group 1 coronaviruses. Thebootstrap values of the subgroup HCoV-NL63/HCoV-229E were 100 for the1a, 1b and S gene. However, for the M and N gene the bootstrap values ofthis subcluster decreased (to 78 and 41 respectively) and a subclustercontaining HCoV-229E, HCoV-NL63 and PEDV becomes apparent. Aphylogenetic analysis could not be performed for the ORF 3 and E genebecause the region varied too much between the different coronavirusgroups or because the region was too small for analysis, respectively.Bootscan analysis by the Simplot software version 2.5²⁹ found no signsof recombination (results not shown).

The presence of a single non-structural protein gene between the S and Egene is noteworthy since almost all coronaviruses have 2 or more ORFs inthis region, with the exception of PEDV and OC43^(30,31). Perhaps mostremarkable is a large insert of 537 nucleotides in the 5′part of the Sgene when compared to HCoV-229E. A Blast search found no similarity ofthis additional 179-amino acid domain of the spike protein to anycoronavirus sequence or any other sequences deposited in GenBank.

TABLE 1 cDNA-AFLP oligonucleotides for virus discovery Oligo SequenceTop strand MSE adaptor CTCGTAGACTGCGTACC (SEQ. ID. NO: 3) Top strand forHinP1 adaptor GACGATGAGTCCTGAC (SEQ. ID. NO: 4) Bottom strand oligo forMSE adaptor TAGGTACGCAGTC (SEQ. ID. NO: 5) Bottom strand oligo for HinP1CGGTCAGGACTCAT adaptor (SEQ. ID. NO: 6) HinPI standard primerGACGATGAGTCCTGACCGC (SEQ. ID. NO: 7) MseI standard primerCTCGTAGACTGCGTACCTAA (SEQ. ID. NO: 8)

TABLE 2 Oligonucleotide for PALM extension of the HCOV-NL68 SequenceOligonucleotide App1i- name, cation, Sequence 5′-3′ JZH2R 1st PCRGCTATCATCACAATGGACNNNNNG (SEQ. ID. NO: 15)

TABLE 3 Nucleotide- and corresponding deduced amino acid sequencesFragment Sequence 163-2GTATTGTTTTTGTTGCTTGTGCCCATGCTGCTGTTGATTCCTTATGTGCAAAAGCTATGACTGTTTATAGCATTGATAAGTGTACTAGGATTATACCTGCAAGAGCTCGGGTTGAGTGTT ATAGTGGCT(SEQ ID NO: 16) 163-2 Replicase polyprotein 1a TranslationIVFVACAHAAVDSLCAKAMTVYSIDKCTRIIPARARVECYSG (SEQ ID NO: 17) 163-4ATGGGTCTAGATATGGCTTGCAAACTTACTACAGTTACCTAACTTTTATTATGTTAGTAATGGTGGTAACAATTGCACTACGGCCGTTATGACCTATTCTAATTTTGGTATTTGTGCTGATGGTTCTTTGATTCCTGTTCGTCC (SEQ ID NO: 18) 163-4 Spike proteinTranslation GSRYGLQNLLQLPNFYYVSNGGNNCTTAVMTYSNFGICADGSLIPVR (SEQ ID NO:19) 163-9 ATGATAAGGGTTTAGTCTTACACACAATGGTAGGCCAGTGATAGTAAAGTGTAAGTAATT(3′-UTR) TGCTATCATAT (SEQ ID NO: 20) 163-10ATGTCAGTGATGCATATGCTAATTTGGTTCCATATTACCAACTTATTGGTAAACAAAAGATAACTACAATACAGGGTCCTCCTGGTAGTGGTAAGTCACATTGTTCCATTGGACTTGGATTGTACTACCCAGGT (SEQ ID NO: 21) 163-10 Replicase polyprotein 1abTranslation VSDAYANLVPYYQLIGKQKITTIQGPPGSGKSHCSIGLGLYYPG (SEQ ID NO: 22)163-11 ATCTAAACTAAACAAAATGGCTAGTGTAAATTGGGCCGATGACAGAGCTGCTAGGAAGAAATTTCCTCCTCCTTCATTTTACATGCCTCTTTTGGTTAGTTCTGATAAGGCACCATATAGGGTCATTCCCAGGAATCTTGTCCCTATTGGTAAGGGTAATAAAGATGAGCAGATTGGTTATTGGAATGTTCAAGAGCGTTGGCGTAT (SEQ ID NO: 23) 163-11 Nucleocapsid proteinSKLNKMASVNWADDRAARKKFPPPSFYMPLLVSSDKAPYRVIPRNLVPIGKGNKDEQIGY WNVQERWR(SEQ ID NO: 24) 163-14ACAAAAATTTGAATGAGGGTGTTCTTGAATCTTTTTCTGTTACACTTCTTGATAATCAAGAAGATAAGTTTTGGTGTGAAGATTTTTATGCTAGTATGTATGAAAATTCTACAATATTGCAAGCTGCTGGTTTATGTGTTGTTTGTGGTTCACAAACTGTACTTCGTTGTGGTGATTGTCTGCGTAAGCCTATGTTGTGCACTAAAT (SEQ ID NO: 25) 163-14 Rsplicase polyprotein1ab TranslationKNLNEGVLESFSVLLDNQEDKFWCEDFYASMYENSTILQAAGLCVVCGSQTVLRCGDCL RKPMLCTK(SEQ ID NO: 26) 163-15AGGGGGCAACGTGTTGATTTGCCTCCTAAAGTTCATTTTTATTACCTAGGTACTGGACCT CATAAGGACCT(SEQ ID NO: 27) 163-15 Nucleocapsid protein TranslationRGQRVDLPPKVHFYYLGTGPHKD (SEQ ID NO: 28) 163-18TAGTAGTTGTGTTACTCGTTGTAATATAGGTGGTGCTGTTTGTTCAAAACATGCAAATTTGTATCAAAAATACGTTGAGGCATATAATACATTTACACAGGCAGGTT (SEQ ID NO: 29) 163-18Replicase polyprotein lab TranslationSSCVTRCNIGGAVCSKHANLYQKYVEAYNTFTQAG (SEQ ID NO: 30)

TABLE 4 Identification of cDNA-AFLP fragments Fragment Identificationbest Blast hit 163-2 replicase polyprotein lab [Human coronavirus 229E]163-4 spike protein [Human coronavirus 229E] 163-9 3′UTR Humancoronavirus 229E 163-10 replicase polyprotein lab [Human coronavirus229E] 163-11 replicase polyprotein lab [Human coronavirus 229E] 163-14replicase polyprotein lab [Human coronavirus 229E] 163-15 nucleocapsidprotein [Human coronavirus 229E] 163-18 replicase polyprotein lab [Humancoronavirus 229E]

TABLE 5 Pairwise nucleotide sequence homologies between the virus of thepresent invention and different corona (like) viruses in percentagessequence identity (%) Fragment BcoV MRV HcoV PEDV TGE SARS IBV Replicase1AB 59.6 61.2 76.7 70.5 64.3 65.8 64.3 163-2 Spike gene 163-4 31.7 26.564.6 48.9 45.4 33.7 25.9 3′UTR 163-9 29.5 34 81.9 53.6 50 31.5 38Replicase 1AB 55.2 57.4 82 73.8 69.4 64.1 65.1 163-10 Nueleocapsid 25.523.8 54.9 51.5 44.6 28.3 27.6 163-11 Replinase 1AB 52.1 52.1 78.7 72.976.3 52.6 58.4 163-14 Nucleocapsid 29.5 35.2 71.8 63.3 60.5 25.8 45163-15 Replicase 1AB 67.2 65.4 72.8 65.4 61.6 68.2 57 163-18

TABLE 6 Pairwise deduced amino acid sequence homologies betweendifferent corona (like) viruses in percentages sequence identity (%)Fragment BCoV MHV HcoV PEDV TGE SARS IBV Replicase 1AB 55.8 53.4 88.379   60.4 67.4 55.8 163-2 Spike gene ND ND 56.2 ND ND ND ND 168-4Replicase 1AB 51.1 53.3 93.3 86.6 80   57.7 55.5 163-10 Nucleocapsid NDND 48.4 ND ND ND ND 163-11 Replicase 1AB 50.7 50.7 86.9 78.2 78.2 46.347.8 163-14 Nucleocapsid ND ND 82.6 ND ND ND ND 168-15 Nucleocapsid 63.863.8 77.7 69.4 69.4 58.3 55.5 163-18 ND = Not Determined

TABLE 7 Oligos for Secific detection of HcoV-163 Primer SequencerepSZ-RT CCACTATAAC (SEQ. ID. NO: 9) repSZ-1 GTGATGCATATGCTAATTTG (SEQ.ID. NO: 10) repSZ-2 TTGGTAAACAAAAGATAACT (SEQ. ID. NO: 11) repSZ-3CTCTTGCAGGTATAATCCTA (SEQ. ID. NO: 12) repSZ-4 TCAATGCTATAAACAGTCAT(SEQ. ID. NO: 13)

TABLE 8 Molecule Features Start End Name Description 287 12439 1a ORF-1a4081 4459 Pfam 01661 9104 10012 3Cl protease 12433 12439 Ribosomueslippery site 12439 20475 1b ORF-1b 14166 14490 Pfam 00680 16162 16965COG1112, Super family DNA and RNA helicase 16237 16914 Pfam 01443 Viralhelicase 20472 24542 2 ORF-2 S(pike)-gene 21099 22619 S1 Pfam 0160122625 24539 S2 Pfam 01601 24542 25219 3 0RF-3 24551 25174 NS3b Pfam03053 25200 25433 4 ORF-4 Pfam 05780, Coronavirus NS4 E (envelope)protein 25442 26122 5 0RF-5 25442 26119 Matrix glycoprotein Pfam 01635M-gene 26133 27266 6 ORF-6 26184 27256 Nucleocapaid Pfam 00937 N-geneVia a −1 frame shift at the ribosome slippery site the 1a ORF isextended to protein of 6729 amino acid residues referred to as 1ab. ORF1a and 1ab encode two polyproteins that are proteolytically converted to16 largely uncharacterized enzymes that are involved in RNA replication(for review seeSnijder, E. J., P. J. Bredenbeek, J. C. Dobbe, V. Thiel,J. Ziebuhr, L. L. Poon, Y. Guan, M. Rozanov, W. J. Spaan, and A. E.Gorbalenya. 2003. Unique and Conserved Features of Genome and Proteomeof SARS-coronavirus, an Early Split-off From the Coronavirus Group 2Lineage. J. Mol. Biol. 331:991-1004).

TABLE 9 Proteins from HcoV-NL63 ORFs ORF Number of AA Mw prediction 1a4060 451364 Polyprotein 1ab 6729 752822 Polyprotein 2 1356 149841 Spike3 225 25658 4 77 9177 Envelope 5 226 25927 Matrix 6 377 42252Nucleocapsid The M_(w) prediction does not take into accountpost-translational modification like glycosylation or cleavage of asignal sequence.

TABLE 10 Amplification oligonucleotides for HCoV-NL65 S, M and Nencoding regions Primer Sequence S1ACAAGTTTGTACAAAAAAGCAGGCTTCAAACTTTCTTGATTTT GCTTGTTTTGCCCC (SEQ ID NO:31) S2 ACCACTTTGTACAAGAAAGCTGGGTCTTGAACGTGGACCTTTTC AAATTCG (SEQ ID NO:32) M1 ACAAGTTTGTACAAAAAAGCAGGCTTCTCTAATAGTAGTGTGCC TCTTTTAGAGG (SEQ IDNO: 33) M2 ACCACTTTGTACAAGAAAGCTGGGTCGATTAAATGAAGCAACTT CTC (SEQ ID NO:34) N1 ACAAGTTTGTACAAAAAAGCAGGCTTCGCTAGTGTAAATTGGGC CGATG (SEQ ID NO:35) N2 ACCACTTTGTACAAGAAAGCTGGGTCATGCAAAACCTCGTTGAC AATTTCTATAATGGC (SEQID NO: 36) The S,M and N complementary sequences are indicated in boldprint. The remainder of the PCR primers is composed of either in-frameattB1 or attB2 sites

TABLE 11 Overall full length genome DNA sequence identity HCoV- HCoV-BCV HC229E IBV SARS TGV NL63 OC43 BCV 100 46 43 54 40 43 95 HC229E 10050 48 53 65 46 IBV 100 43 46 48 43 SARS 100 40 43 53 TGV 100 55 40 HCoV-100 43 NL63 OC43 100 Overall DNA sequence identity percentages ofHCoV-NL63 compared to other coronaviruses. From the SimPlot graph (FIG.7), comparing HCoV-NL63 (query) with SARS associated coronavirus andHCoV-229E, can be deduced that local sequence identity never exceeds 85%

TABLE 12 Overall DNA sequence identity Spike encoding region OC48 NL63229E SARS OC43 100 46 40 44 NL63 100 59 38 229E 100 41 SARS 100

TABLE 13 Overall DNA sequence identity in 5′UTR OC43 NL63 229E SARS OC43100 36 34 48 NL63 100 74 33 229E 100 34 SARS 100

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1

cDNA-AFLP allows amplification of nucleic acids without any priorsequence information.

Culture supernatants from CPE-positive and uninfected cells aresubjected to the cDNA-AFLP procedure. Amplification products derivedfrom the CPE-positive culture which are not present in the uninfectedcontrol sample are cloned and sequenced.

FIG. 2

LLC-MK2 cells infected with HCoV-NL168.

Panel A and B are unstained cells while panel C and D are stained withhaematoxilin eosin. The typical CPE of HCoV-NL163 is shown in panel Aand C. The control uninfected LLC-MR cells are shown in panel B and D.

FIG. 3

VD-cDNA-AFLP PCR products visualized by Metaphors agarose gelelectrophoreses.

The PCR products of 1 (HinP I-G and Mse I-A) of 16 primer paircombinations used during the selective amplification step. Lanes 1 and2: duplicate PCR product of virus culture NL163; lanes 5 and 6 controlsupernatant of LLC-MK cells and in lane 7 and 8 the negative PCRcontrol. Lanes M: 25 bp molecular weight marker (InVitrogen). The arrowindicates a new coronavirus fragment that was excised out of gel andsequenced.

FIG. 4

Phylogenetic analysis of the HCoV-163 sequences. G1, G2 and G3 denotethe group 1, group 2 and group 3 coronavirus clusters. The Genbankaccession number of the used sequences are: MHV (mouse hepatitis virus):AF201929; HCoV-229E: AF304460; PEDV (porcine epidemic diarrhea virus):AF353511; TGEV (transmissible gastroenteritis virus): AJ271965;SARS-CoV: AY278554; IBV (avian infectious bronchitis virus):NC_(—)001451; BCoV (bovine coronavirus): NC_(—)003045; FCoV (felinecoronavirus): Y13921 and X80799; CCoV (canine coronavirus): AB105373 andA22732; PRCoV (porcine respiratory coronavirus): M94097; FIPV (felineinfectious peritonitis virus): D32044. Position of the HCoV-163fragments compared to HCoV-229E (AF304460): Replicase 1AB gene:15155-15361, 16049-16182, 16190-16315, 18444-18550, Spike gene:22124-22266; Nucleocapsid gene: 25667-25882 and 25887-25957; 3′UTR:27052-27123. Branch lengths indicate the number of substitutions persequence.

FIG. 5

schematic representation of Coronavirus and the location of the163-fragments listed in table 3.

FIG. 6

Restriction map of HCoV-NL63′

Complete 27553 nt cDNA derivative of the ssRNA genome. Open readingframes (ORF) are depicted as numbered black arrows and the identified(PFAM) domains within these ORFs are indicated as gray boxes.

FIG. 7

Simplot analysis HcoV NL63 and other human Coronaviruses The gap in thecomparison of HCoV NL63 to SARS, HCoV-OC43 and HCoV-229E is cause by aunique 537 in-frame insertion in the Spike protein encoding ORF (seeelsewhere herein). Sigmaplot analysis is described in Lole, R. S., R C.Bollinger, R. S. Paranjape, D. Gadkari, S. S. Kulkarni, N. G. Novak, R.Ingersoll, H. W. Sheppard, and S. C. Ray. 1999. Full-length humanimmunodeficiency virus type 1 genomes from subtype C-infectedseroconverters in India, with evidence of intersubtype recombination. J.Virol. 73:152-160.

FIG. 8

Expression constructs for HCoV-NL63 Spike and Matrix protein

Expression of a His and StrepII tagged Spike fusion protein can beinduced by addition of IPTG to the bacterial growth medium. ThroughattB1/B2-mediated recombination, the S gene insert can be transferred toother commercially available expression vectors, facilitating proteinproduction in other hosts.

Through an identical cloning procedure as for pGP7S, a Gatewaycompatible expression vector for HCoV-NL63 M-gene can be constructed.The plasmid directs IPTG inducible production of N and C-terminallyaffinity tagged Matrix fusion protein, allowing selective recovery offull-length fusion protein.

FIG. 9

Recombination site NL63-229E (SEq. ID. NO: 38)

NL63-derived sequences are in underlined bold black print and the 229Ederived sequences are in gray bold print.

FIG. 10

Restriction map cDNA Clone NL63/229E hybrid The NL63 derived part isindicated as gray boxes and the 229E-derived region is indicated as aline. The junction between the two genomes is indicated by thesuccession of the two black arrows marked 1b′ and ORF-1b indicating thehybrid 1b ORF.

A second chimeric genome was generated by a reciprocal recombinationfusing nucleotide 19653 of HCoV-NL63 to nucleotide 20682 of HCoV-OC43again creating a hybrid ORF 1b giving rise to a hybrid 1ab replicasepolyprotein. Recombination occurred within the conserved sequenceAATTATGG (SEQ. ID. NO: 37).

FIG. 11 Recombination site NL63/OC43 hybrid (SEQ. ID. NO: 39).

Again, NL63-derived region is in bold black underlined print and theOC43 derived sequences are in gray bold print. The resulting cDNArestriction map is depicted in FIG. 12

FIG. 12

Restriction map recombinant NL63/OC43 genome.

The NL63-derived part is indicated as gray boxes and the recombinationsite is depicted as the between the black arrows 1b′ and 1b.

FIG. 13

Similarity plot deduced protein alignments of ORF1b from HCoV-NL63,HCoV-229E, HCoV-OC43 and the two hybrids NL63/229E and NL63/OC43.

FIG. 14

Green fluorescent protein expressing HcoV-NL63 derivative. Functionalequivalent NL63/4GFP carries an in-frame C-terminal fusion of the Eprotein (ORF4) with a human codon optimised Green Fluorescent Protein(EGFP, Stratagene). Infected cells appear fluorescent after excitationof the 4-EGFP fusion protein. HCoV-NL63 can be used to elucidate theprocess of viral; infection and the translation of the polycistronicsub-genomic messengers.

FIG. 15

Restriction map of functional derivative NL63D2052021011.

This deletion derivative of NL63 lacks most of the insertion at theN-terminal end of the Spike protein. By deleting nucleotides 20520-21011the unique domain is removed while retaining the predicted secretorysignal sequence (Nielsen, H., J. Engelbrecht, S. Brunak, and G. VonHeijne. 1997. Identification of prokaryotic and eukarotic signalpeptides and prediction of their cleavage sites. Protein Eng 10:1-6).

FIG. 16

Sequence variation in HCoV-NL63 from additional patient samples Directsequencing of both strands of RT-PCR products from 6 patient samplesrevealed the presence of polymorphisms in the ORF 1a region. REF (SEQ.ID. NO: 40); 223 B (SEQ. ID. NO: 41); 246 B (SEQ. ID. NO: 42); 248 B(SEQ. ID. NO: 43); 251 B (SEQ. ID. NO: 44); 466 B (SEQ. ID. NO: 45); 496B (SEQ. ID. NO: 46)

FIG. 17

HCoV-NL63 specific and generic human Coronavirus detection probes.Coronavirus polymerases generate several sub-genomic RNAs. The frequencyof S. E, M and N protein encoding cDNA clones in the sequencing libraryof HCoV-NL63 and SARS (Snijder, E. J., P. J. Bredenbeek, J. C. Dobbe, V.Thiel, J. Ziebuhr, L. L. Poon, Y. Guan, M. Rozanov, W. J. Spaan, and A.E. Gorbalenya. 2003). Unique and conserved features of genome andproteome of SARS-coronavirus, an early split-off from the coronavirusgroup 2 lineage. J. Mol. Biol. 331:991-1004). Northern blot datademonstrate a high abundance of these sub-genomic RNAs in infectedcells. Consequently, these genes are attractive targets for diagnostictests.

Since the genomic and sub-genomic RNAs possess identical 3′ends, probescontaining the N gene would hybridise to all of them (Table 8).

Through alignment of the full-length sequences of all humanCoronaviruses a conserved region in ORF1b was identified, allowing theirdetection with a nested RT-PCR assay. Oligo NL63NF1 (SEQ. ID. NO: 47);Oligo NL63NR1 (SEQ. ID. NO: 48); Oligo NL63NF2 (SEQ. ID. NO: 49); OligoNL63NR2 (SEQ. ID. NO: 50)

FIG. 18

Generic Coronavirus detection primers. Oligo COR1F (SEQ. ID. NO: 51);Oligo COR1R (Seq. ID. NO: 52), Oligo COR2F (SEQ. ID. NO: 53); OligoCOR2R (SEQ. ID. NO: 54)

FIG. 19

Nucleotide sequence an HcoV_NL63 (SEQ. ID. NO: 55)

FIG. 20

ORF 1a, replicase enzyme complex of an HcoV_NL63 (SEQ. ID. NO: 56)

FIG. 21

ORF 1 ab replicase polyprotein of an HcoV_NL63 (SEQ. ID. NO: 57).Adenosine diphosphate-ribose 1′-phosphate (SEQ. ID. NO: 58). 3CI^(Pro)Coronavirus polyprotein processing endoprotease (SEQ. ID. NO: 59); RNAdependent RNA polymerase (pfam00680) (SEQ. ID. NO: 60); Exon 3′ to 5′Exonuclease and helicase (SEQ. ID. NO: 61); XendoU (homolog of)polyU-specific endoribonuclease (SEQ. ID. NO: 62); 2′-O-MT2:S-adenosylmethionine-dependnet ribose 2′-orthomethyltransferase (SEQ.ID. NO: 63

FIG. 22

The spike protein (ORF3) contains an N-terminal secretory signalsequence of 16 AA (indicated on the first line of the continuoussequence listed below). (Nielsen, H., J. Engelbrecht, S. Brunak, and G.Von Heijne. 1997. Identification of prokaryotic and eukaryotic signalpeptides and prediction of their cleavage sites. Protein Eng 10:1-6)

FIG. 28

ORF-4 Coronavirus_NS4, Coronavirus non-structural protein 4. This familyconsists of several non-structural protein 4 (NS4) sequences or smallmembrane protein.

ORF-5. This family consists of various coronavirus matrix proteins thatare transmembrane glycoproteins. The M protein or E1 glycoprotein isimplicated in virus assembly. The E1 viral membrane protein is requiredfor formation of the viral envelope and is transported via the Golgicomplex. The matrix protein is predicted to contain an N-terminalsecretory signal sequence (indicated in the first part of the continuoussequence) (Nielsen, H., J. Engelbrecht, S. Brunak, and G. Von Heijne.1997. Identification of prokaryotic and eukaryotic signal peptides andprediction of their cleavage sites. Protein Eng 10:1-6.)

ORF-6 Pfam 00937, Coronavirus nucleocapsid protein. Structural proteinforming complexes with the genomic RNA

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1. An isolated and/or recombinant nucleic acid sequence comprising anucleic acid sequence at least 95% homologous to the entire sequence ofSEQ ID NO:
 55. 2. An isolated and/or recombinant nucleic acid sequencecomprising a stretch of 100 consecutive nucleotides of the isolatedand/or recombinant nucleic acid sequence of claim
 1. 3. An isolatedand/or recombinant nucleic acid sequence according to claim 1, encodinga proteinaceous molecule.
 4. An isolated and/or recombinant viruscomprising a nucleic acid sequence at least 95% homologous to the entiresequence of SEQ ID NO:
 55. 5. The isolated and/or recombinant virus ofclaim 4, wherein said isolated and/or recombinant virus is capable ofinducing a HCoV-NL63- related disease in a subject.
 6. A vectorcomprising the isolated and/or recombinant nucleic acid sequence ofclaim
 1. 7. A composition comprising the isolated and/or recombinantvirus of claim 4 and a pharmaceutically acceptable carrier or excipient.8. An isolated cell comprising the isolated and/or recombinant virus ofclaim
 4. 9. The cell of claim 8, wherein said cell is a primate cell.10. The cell of claim 8, wherein said cell is a kidney cell.
 11. Thecell of claim 9, wherein said cell is a monkey cell.
 12. A gene deliveryvehicle comprising the isolated and/or recombinant nucleic acid sequenceof claim
 1. 13. The isolated and/or recombinant virus of claim 4,wherein said isolated and/or recombinant virus has been attenuated. 14.An isolated and/or recombinant nucleic acid sequence comprising theentire sequence of SEQ ID NO:
 55. 15. An isolated and/or recombinantnucleic acid sequence comprising a stretch of 100 consecutivenucleotides of the isolated and/or recombinant nucleic acid sequence ofclaim
 14. 16. An isolated and/or recombinant nucleic acid sequenceaccording to claim 14, wherein said sequence encodes a proteinaceousmolecule.
 17. An isolated and/or recombinant virus comprising thenucleic acid sequence of SEQ ID NO:
 55. 18. The isolated and/orrecombinant virus of claim 17, wherein said isolated and/or recombinantvirus is capable of inducing a HCoV-NL63- related disease in a subject.19. A vector comprising the isolated and/or recombinant nucleic acidsequence of claim
 14. 20. A composition comprising the isolated and/orrecombinant virus of claim 17, and a pharmaceutically acceptable carrieror excipient.
 21. An isolated cell comprising the isolated and/orrecombinant virus of claim
 17. 22. The cell of claim 21, wherein saidcell is a primate cell.
 23. The cell of claim 21, wherein said cell is akidney cell.
 24. The cell of claim 22, wherein said cell is a monkeycell.
 25. A gene delivery vehicle comprising the isolated and/orrecombinant nucleic acid sequence of claim
 14. 26. The isolated and/orrecombinant virus of claim 17, wherein said isolated and/or recombinantvirus has been attenuated.